A Bio-Tech Study in Gene Silencing and RNA Interference

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A Bio-Tech Study in Gene Silencing and RNA Interference

Stace Acala

Amy Demins

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NIH Curriculum: Silence of the Genes – A Bio-Tech Study in Gene Silencing and RNA Interference

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Authors: Stace Alcala – Mulberry High School and Amy Demins – Oviedo High School

This curriculum was developed as a part of Biomedical Explorations: Bench to Bedside which

was supported by the National Center for Research Resources and the Division of Program

Coordination, Planning, and Strategic Initiatives of the National Institutes of Health through

Grant Number R25RR023294. Additional support provided by the University of Florida (UF) and

the UF Center for Precollegiate Education and Training.

The content is solely the responsibility of the author and does not necessarily represent the

official views of the National Center for Research Resources or the National Institutes of Health.

Additional information regarding the Bench to Bedside project is available at

http://www.cpet.ufl.edu/bench.

Please direct inquiries to the Center for Precollegiate Education and Training at

[email protected].

Last updated: 9/13/2012

©2012 University of Florida

Center for Precollegiate Education and Training

PO Box 112010 • Yon Hall, Room 331

Gainesville, FL 32611

Phone 352.392-2310• Fax 352.392-2344

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CONTENTS

Author’s Note and Introduction .................................................................................................................................... 6

Lesson Summaries ......................................................................................................................................................... 7

Lesson Sequencing Guide .............................................................................................................................................. 9

Lesson Vocabulary: ...................................................................................................................................................... 10

Next Generation Sunshine State Standards - NGSSS ................................................................................................... 13

Background Information .............................................................................................................................................. 16

Silkworm Husbandry .................................................................................................................................................... 17

LESSON ONE: Eat – “Sleep” – and Be Quiet! ........................................................................................................... 21

Teacher Pages: Primary Protein Modeling – Teacher Answers ................................................................................... 25

Student Pages: Primary Protein Modeling – Student Worksheet ............................................................................... 27

Teacher Pages: Amino Acid Bingo: “Scoring Card” – Form A ...................................................................................... 29

Teacher Pages: Amino Acid Bingo: “Scoring Card” – Form B ....................................................................................... 30

Teacher Pages: Amino Acid Bingo: “Scoring Card” – Form C ....................................................................................... 31

Teacher Pages: Amino Acid Bingo: Instructor Directions ............................................................................................ 32

Teacher Pages: Introduction Powerpoint – Silkworm Life-Cycle - Agenda .................................................................. 33

Teacher Pages: Universal Codon Chart ........................................................................................................................ 35

Teacher Pages: Example of protein modeling card (1 of 10) ...................................................................................... 36

LESSON TWO: Who Put the “i” in my RNA? ................................................................................................................. 37

Teacher Pages: Jigsaw Activity Group Assignments .................................................................................................... 40

Student Pages: Jigsaw Activity Group Assignments .................................................................................................... 52

LESSON THREE: “I Said – BE QUIET!!!” ....................................................................................................................... 63

Teacher Pages: Discussion Questions from NOVA Video and Research PowerPoint Key ........................................... 66

Student Pages: Discussion Questions from NOVA Video and Research PowerPoint ................................................. 67

Teacher Pages: Pipetting by Design ............................................................................................................................ 68

LESSON FOUR: Let’s See What’s In There .................................................................................................................... 69

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Student Pages: Silkworm DNA Extraction / PCR .......................................................................................................... 72

Student Pages: Silkworm DNA Gel Electrophoresis ..................................................................................................... 73

Student Pages: Silkworm Protein Extraction and Electrophoresis .............................................................................. 74

Teacher Pages: PowerPoint on Silkworm PCR / Electrophoresis ................................................................................. 75

LESSON FIVE: Who to Choose? – Speak Up!! ............................................................................................................. 77

Student Page: Use as a WebQuest (See Appendix #1) ................................................................................................ 81

Student Pages: Silkworm DNA Gel Electrophoresis Worksheet .................................................................................. 83

LESSON SIX: The Proposal ............................................................................................................................................ 85

LESSON SEVEN: Were you Quiet or Not? That, is the Questions ................................................................................ 89

Student Pages: Formal Lab Write-Up – Day 2.............................................................................................................. 91

Student Pages: Data Collection Worksheet ................................................................................................................. 93

Appendix #1 ................................................................................................................................................................. 95

Appendix #2 Exit Card .................................................................................................................................................. 97

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Author’s Note and Introduction I began studying Bombyx mori (the common silkworm) in 2008 with a classroom project involved in simply rearing the species to generate interest in zoology. Although it was challenging to find their exclusively preferred food source (mulberry leaves), the larva were easy to care for and quickly developed into three inch “caterpillars”. We anticipated some unique method of getting the “worms” to spin the highly sought silk, yet, they accomplished the feat quite on their own. The larva merely crawled to the edges of their habitat seeking two perpendicular walls and within two days was woven tightly into their silky white capsules. Three weeks later the moths crawled from their metamorphic isolation chambers and began their last days seeking a mate. We had read that they not only died within a week, but did not eat and could not fly! Alas, their only desire was to find a mate and produce a fine clutch of approximately 300 eggs. Students and instructor were fascinated with the saga and thus began a seasonal classroom project. As the years have passed the questions involved with silkworms have accumulated. Such questions as: “what makes them change from larva to pupa?”, “how do they know when they are supposed to cocoon?”, “is the DNA of a moth the same as the larva?” among others. Last year we answered some of those questions with the introduction of biotechnology to our classroom. The students extracted both protein and DNA from each of the metamorphic stages of classroom reared silkworms. In this study the students used two known gene sequences for both silk and membrane transport proteins. With the aid of manufactured primers, the students took the extracted DNA and performed the Polymerase Chain Reaction (PCR) upon the samples. This technology induces amplification of the genes to many 1000’s of copies. With the completion of this process the students then learned the process of gel-electrophoresis and segregated the gene “bands” through a porous agarose gel slab. Like a CSI episode, the students learned how to produce a DNA “fingerprint”. What they found answered one nagging question, “is the DNA of moth the same as a larva?”, and of course they found the answer to be “yes”! The excitement this investigation led me to the conclusion that we were on to something and it needed to continue. The students were becoming more interested in science. This previous study has led to the current curriculum development. This time we are going to include most of the previous lessons, yet, our focus will be on the newest of technologies, that is, silencing the production of proteins. The technique, called gene silencing incorporates a laboratory designed RNA called RNAi (i for interference). In a nutshell, the technology uses a designer RNAi and places it into the host’s genome causing cellular “police” to deactivate both the foreign gene sequence and the host gene sequence. In effect, the protein does not get made. In our case, we will be using RNAi’s to cease the production of the known silk gene we used last year. It is our intention to “feed” (literally) the RNAi to the silkworm larva in the hopes to shut off the protein that it makes silk with. Theoretically these little guys will not be able to cocoon when it is time. Although this sounds quite harsh (as this is the main pathway to their adult nirvana) the worms should be able to pupate anyway. Yet, we are mostly interested in seeing if the technology can be done with the silkworms. Stay tuned, we are about to begin the journey into “Silence of the Genes”

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Lesson Summaries

LESSON ONE: Eat, Sleep, and Be…. QUIET! The students will be introduced to the concept of gene silencing with a short PowerPoint that highlights cellular and molecular mechanisms (transcription and translation), as well as a generalized view of the silkworm life cycle. A fundamental understanding of these mechanisms is crucial in the development of the unit. A 20 question pre-investigative assessment will be given to produce a knowledge baseline. Two activities (amino acid bingo and primary protein modeling) are intended to reinforce the concepts as well as produce a heightened interest.

LESSON TWO: Who Put the “i” in My RNA? Using a jigsaw approach, students will learn about the fundamentals of RNA interference. In Part 1, students are divided into 6 groups: Argonautes, C. elegans, Dicers, RISC, RNAis, and siRNAs. Each group is given one or two resources and a set of questions for each of the resources. When they have completed Part 1, they will then switch into of four groups for Part 2. Part 2 has a student worksheet with more general questions about RNAi. As a group, the students need to complete the Part 2 worksheet. For Part 3, students will return to their original group and draw a diagram of the process of RNA interference. They will work out their ideas on white boards, and then transfer their final diagram to the Part 3 worksheet.

LESSON THREE: “I Said – Be Quiet!” The students will be introduced to the research laboratory facilities and personnel of Dr. Gregory Schultz. A PowerPoint will highlight pictures of laboratory equipment/space and a generalization of the corneal tissue repair experimentation currently in progress dealing with gene silencing. In addition, the students will watch a Nova video that takes them on a short journey of the history of gene silencing and its discovery within petunias. Finally, hands-on review of biotechnology techniques will prepare the class for Lesson 4’s DNA extraction and PCR set-up for silk gene amplification.

LESSON FOUR: Let’s See What’s in There Students will follow instructions on DNA extraction for each of 3 samples of silkworm metamorphic stages (egg, larva, pupa, adult). PCR will be used to amplify silk and membrane genes extracted from each of the metamorphic samples. These extractions will then be electro-phoresed and used as a control against the DNA genome that will be hypothetically “silenced” (during our analysis dates).

LESSON FIVE: Who To Choose? – SPEAK UP!! Students will use web-based information (webquest) to learn the methodologies associated with RNAi selection. A step-wise process is also found on a student handout that will guide them in the process of selection (even without the web sites). However, all RNAi selections must be performed using the BLAST protocols and sequence comparisons. In addition, the students will use the amplified genes from the previous lesson’s DNA extractions and run gel-electrophoresis to segregate gene “bands”. Gels will be run at the beginning of the lesson to allow web use during “down time” of the electrophoresis units. Finally, students will apply the ingestible RNAi’s to the silkworm leaves to gain transfection of RNAi into the host’s cellular spaces.

LESSON SIX: The Proposal The purpose of this lesson is to introduce students to the process of writing a proposal for scientific research. They will then present their proposals to the class (scientific community) using Glog they have created on Glogster.com (http://edu.glogster.com/). The artifact of the lesson is that the students are University of Florida graduate students interested in studying RNA interference. Students will need to use the knowledge they have gained in the previous lessons to develop an outline of an experiment using RNAi. Students will need to first identify the problem, how they are going to study the problem, what model organism to use, and possible applications of their results.

http://edu.glogster.com/

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LESSON SEVEN: Were You Quiet or Not? That is the Question Students will finalize the silkworm unit with collection of data that includes quantitative measurements and visual assessments of cocoon mass/size based on a mean of similarly altered experimental group cocoons vs control group cocoon means. A “final lab write-up” will be required and used for assessment – criteria for write-up will be provided to each student with due dates.

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Lesson Sequencing Guide (A Prescribed Pacing Guide – 50 minute periods)

Since the classroom teacher knows his or her students best, the teacher should decide on the sequencing of the lessons. This table demonstrates a suggested pacing guide that can be used when planning to use this curriculum. All lessons are based on a 50 minute class session:

DAY 1 DAY 2 DAY 3 DAY 4 DAY 5

WEEK 1

LESSON 1

EAT, SLEEP

AND BE QUIET!

LESSON 2

WHO PUT THE “i” IN MY RNAi

LESSON 3

I SAID - “BE QUIET!”

LESSON 4

LET’S SEE WHAT’S IN

THERE

LESSON 5

WHO TO CHOOSE?

SPEAK UP!!

WEEK 2

LESSON 5

WHO TO CHOOSE?

SPEAK UP!!

LESSON 6

THE PROPOSAL

LESSON 6

THE PROPOSAL

LESSON 6

THE PROPOSAL

LESSON 7 *

WERE YOU QUIET OR

NOT? (THAT IS THE QUESTION)

WEEK 3

LESSON 7 *

WERE YOU QUIET OR

NOT? (THAT IS THE QUESTION)

COMPLETE ANY

DATA FOR WRITE-UP

COMPLETE ANY

DATA FOR WRITE-UP

COMPLETE ANY

DATA FOR WRITE-UP

FINAL LAB WRITE UP DUE

*Lesson 7 is the final lesson for analysis, it may take place several days after the other lessons as the larva will be morphing into new instar stages prior to cocooning.

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Lesson Vocabulary: Adjustable Micropipetting: An adjustable micropipettor is essentially a precision pump fitted with a disposable tip. The volume of air space in the barrel is adjusted by screwing the plunger farther in or out of the piston, and the volume is displayed on a digital readout. Depressing the plunger displaces the specified volume of air from the piston; releasing the plunger creates a vacuum, which draws an equal volume of fluid into the tip. The withdrawn fluid is then expelled by depressing the plunger again. Agarose: A gelatinous substances derived from the structures of red algae, used as a nutrient media for culturing bacterial plates and as a porous media for gel-electrophoresis (due to its relatively neutral charge). Amino Acids: An organic molecule containing an amino group (-NH2), a carboxyl (-COOH) group, and a variable side chain (R group) that distinguishes the amino acid. Proteins are synthesized from amino acids. Amplification: An increase in the number of copies of a gene in a cell, resulting in an elevation in the level of the RNA or protein encoded for by the gene and a corresponding amplification of the phenotype that the gene confers on the cell. Drug resistance in cancer cells is linked to amplification of the gene that prevents absorption of the chemotherapeutical agent by the cell. Annealing: To recombine (nucleic acids) at low temperature after separating by heat. Argonaute: Protein that is the main component of the RNA induced silencing complex (RISC) and aids in the recognition and cleavage of mRNA during RNAi. BLAST: The Basic Local Alignment Search Tool (BLAST) finds regions of local similarity between sequences. The program compares nucleotide or protein sequences to sequence databases and calculates the statistical significance of matches. BLAST can be used to infer functional and evolutionary relationships between sequences as well as help identify members of gene families. http://blast.ncbi.nlm.nih.gov/Blast.cgi Bombyx mori: Scientific name of the most common form of silkworm, proposed to originate in China Enzymes: Any of numerous proteins produced in living cells that accelerate or catalyze chemical reactions. Complete and Incomplete Metamorphosis: Changes that occur within the life-cycle of many organisms. The terms typically refer to entomological organisms. Complete metamorphosis: egg, larva, pupa, adult. Incomplete metamorphosis: egg, nymph, and adult Codon: a triplet sequence of nucleotides that directs the tRNA’s ”anticodon” to acquire select amino acids for protein production. Cornea: a transparent “window” into the eye. Recognized as a-vascular, the cornea is composed of epithelial, stromal, and endothelial tissues. It is the first surface of light refraction in visual acuity. Denaturation: A process in which the structure of nucleic acid is disrupted, such as the dissociation of a double stranded DNA into a single stranded state by heating. Dicer: A nuclease that cleaves double-stranded RNAs. Dicer processes long dsRNA into siRNA or miRNA or 21-23 nucleotides. E–Gel cassette: a premade agarose gel slab held within a plastic “cassette” that can be inserted into a compact and movable power source. Extension: The addition of nucleotides to starter primers on a desired gene for amplification.

http://blast.ncbi.nlm.nih.gov/Blast.cgi

http://www.biology-online.org/dictionary/Nucleic_acid

http://www.biology-online.org/dictionary/Dissociation

http://www.biology-online.org/dictionary/DNA

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Gel – electrophoresis: Agarose gel electrophoresis is an easy way to separate DNA fragments by their sizes and visualize them. It is a common diagnostic procedure used in molecular biological labs. Gene Silencing: A method of regulating gene expression using components that offer “missense” such as RNAi and siRNA – any of which interrupts the general formation of the typical protein formation. Helicase: The enzyme responsible for “unzipping” DNA during replication and RNA formation. Macular Degeneration: Loss of centralized vision often due to detachment of the retina from the choroid. Dry form of degeneration forms from accumulation of drusen (cellular debris) detaching retina, wet form caused by accumulating blood vessels which also detach retina. mRNA: An abbreviated expression for Messenger Ribonucleic Acid. This genetic sequence is responsible for “coding” a gene within the DNA for the instruction of constructing proteins within the cell’s cytoplasm at a ribosome. Microcentrifuge: An apparatus consisting essentially of a compartment spun about a central axis to separate contained materials of different densities, or to separate colloidal particles suspended in a liquid. miRNA: Micro-RNAs (miRNAs) are single-stranded RNAs of 21-23 nt. Similar to siRNAs, miRNAs can silence gene activity via destruction of homologous mRNA. NCBI: National Center for Biotechnology Information – advances science and health by providing genomic information. http://www.ncbi.nlm.nih.gov/ Poly-A tail: Polyadenylation is the addition of a poly(A) tail to an RNA molecule. The poly(A) tail consists of multiple adenosine monophosphates; in other words, it is a stretch of RNA that has only adenine bases. In eukaryotes, polyadenylation is part of the process that produces mature messenger RNA (mRNA) for translation. Polymerase: The enzyme responsible for DNA replication and RNA formations. Proteins: A linear polymer built from approximately 20 different amino acid types. The type and sequence of these amino acids are specified by the DNA, this sequence also determines shape and function of the protein. Ribozymes: An RNA molecule with a tertiary structure that often performs catalytic (enzymatic) functions. It is known to self-cleave which consumes it’s own structure. Ribozyme means: ribonucleic acid enzyme RISC: The RNA induced silencing complex (RISC) is composed of siRNA (or miRNA), Argonaute protein, and other proteins that catalyze cleavage of the mRNA target. RNAi: RNA interference (RNAi) is the process of using small pieces of double-stranded RNA to reduce the activity of specific genes. The process occurs naturally in many organisms and is now commonly used in basic research. siRNA: Small interfering RNA (siRNA) is 21-23 nt double-stranded RNA molecules formed from the cleavage of long dsRNA by dicer. The siRNA guides the cleavage and degradation of its target mRNA. tRNA: An abbreviated expression for Transfer Ribonucleic Acid. This genetic sequence is responsible for the “gathering” of amino acids within the cell’s cytoplasm. Thermocycler: a laboratory apparatus used to amplify segments of DNA via the polymerase chain reaction (PCR) process.

http://www.ncbi.nlm.nih.gov/

http://en.wikipedia.org/wiki/RNA

http://en.wikipedia.org/wiki/Adenosine_monophosphate

http://en.wikipedia.org/wiki/Adenine

http://en.wikipedia.org/wiki/Eukaryote

http://en.wikipedia.org/wiki/Messenger_RNA

http://en.wikipedia.org/wiki/Translation_(biology)

http://en.wikipedia.org/wiki/Laboratory

http://en.wikipedia.org/wiki/DNA

http://en.wikipedia.org/wiki/Polymerase_chain_reaction

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Transcription: The process of RNA production within the nucleus using DNA as a “template” to copy a specific location within the DNA (typically a gene). The DNA is “transcribed” from the 5’3’ using nucleotides. Transilluminator: The passing of a light through the walls of a body part or organ to facilitate internal inspection. Translation: A “decoding” of the mRNA (created in transcription) at a ribosome within the extranuclear space. The process produces primary structure proteins that can be later modified to perform an assortment of structural/physiological functions. 5’ UTR: The five prime UnTranslated Region (5' UTR), can contain elements for controlling gene expression by way of regulatory elements. It begins at the transcription start site and ends one nucleotide (nt) before the start codon (usually AUG) of the coding region. In prokaryotes, the 5' UTR usually contains a ribosome binding site (RBS), also known as the Shine Dalgarno sequence (AGGAGGU).

http://en.wikipedia.org/wiki/Gene

http://en.wikipedia.org/wiki/Regulatory_element

http://en.wikipedia.org/wiki/Transcription_start_site

http://en.wikipedia.org/wiki/Nucleotide

http://en.wikipedia.org/wiki/Start_codon

http://en.wikipedia.org/wiki/Coding_region

http://en.wikipedia.org/wiki/Prokaryotes

http://en.wikipedia.org/wiki/Ribosome_binding_site

http://en.wikipedia.org/wiki/Shine_Dalgarno

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Next Generation Sunshine State Standards - NGSSS

Benchmark Lesson

1 2 3 4 5 6 7

SC.912.L.14.2 Relate structure to function for the components of plant and animal cells. Explain the role of cell membranes as a highly selective barrier (passive and active transport).

X X X X

SC.912.L.14.3 Compare and contrast the general structures of plant and animal cells. Compare and contrast the general structure of prokaryotic and eukaryotic cells

X X X X

SC.912.L.14.6 Explain the significance of genetic factors, environmental factors, and pathogenic agents to health from the perspectives of both individual and public health.

X X X X X

SC.912.L.14.50 Describe the structure of vertebrate sensory organs. Relate structure to function in vertebrate sensory systems.

X

SC.912.L.15.15 Describe how mutation and genetic recombination increase genetic variation.

X X X

SC.912.L.16.2 Discuss observed inheritance patterns caused by various modes of inheritance, including dominant, recessive, codominant, sex-linked, polygenic, and multiple alleles.

SC.912.L.16.3 Describe the basic process of DNA replication and how it relates to the transmission and conservation of the genetic information.

X X X X

SC.912.L.16.4 Explain how mutations in the DNA sequence may or may not result in phenotypic change. Explain how mutations in gametes may result in phenotypic changes in offspring.

X X X X X

SC.912.L.16.5 Explain the basic processes of transcription and translation, and how they result in the expression of genes.

X X X X X X

SC.912.L.16.6 Discuss the mechanisms for regulation of gene expression in prokaryotes and eukaryotes at transcription and translation level

X X X X X

SC.912.L.16.7 Describe how viruses and bacteria transfer genetic material between cells and the role of this process in biotechnology.

X X X X

SC.912.L.16.8 Explain the relationship between mutation, cell cycle, and uncontrolled cell growth potentially resulting in cancer.

X

SC.912.L.16.9 Explain how and why the genetic code is universal and is common to almost all organisms.

X X X X X

SC.912.L.16.10 Evaluate the impact of biotechnology on the individual, society and the environment, including medical and ethical issues.

X X X X X X X

SC.912.L.16.11 Discuss the technologies associated with forensic medicine and DNA identification, including restriction fragment length polymorphism (RFLP) analysis.

X X

SC.912.L.16.12 Describe how basic DNA technology (restriction digestion by endonucleases, gel electrophoresis, polymerase chain reaction, ligation, and transformation) is used to construct recombinant DNA molecules (DNA cloning).

X X X X X

SC.912.L.18.1 Describe the basic molecular structures and primary functions of the four major

X

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Benchmark Lesson

1 2 3 4 5 6 7

categories of biological macromolecules.

SC.912.L.18.4 Describe the structures of proteins and amino acids. Explain the functions of proteins in living organisms. Identify some reactions that amino acids undergo. Relate the structure and function of enzymes.

X X X

SC.912.L.18.11 Explain the role of enzymes as catalysts that lower the activation energy of biochemical reactions. Identify factors, such as pH and temperature, and their effect on enzyme activity.

X

SC.912.N.1.1 Define a problem based on a specific body of knowledge, for example: biology, chemistry, physics, and earth/space science, and do the following:

1. pose questions about the natural world, 2. conduct systematic observations, 3. examine books and other sources of information to see what is already known, 4. review what is known in light of empirical evidence, 5. plan investigations, 6. use tools to gather, analyze, and interpret data, 7. pose answers, explanations, or descriptions of events, 8. generate explanations that explicate or describe natural phenomena (inferences), 9. use appropriate evidence and reasoning to justify these explanations to others, 10. communicate results of scientific investigations, and

11. evaluate the merits of the explanations produced by others.

X X X

SC.912.N.1.2 Describe and explain what characterizes science and its methods.

X X

SC.912.N.1.3 Recognize that the strength or usefulness of a scientific claim is evaluated through scientific argumentation, which depends on critical and logical thinking, and the active consideration of alternative scientific explanations to explain the data presented.

X X

SC.912.N.1.4 Identify sources of information and assess their reliability according to the strict standards of scientific investigation.

X X X

SC.912.N.1.5 Describe and provide examples of how similar investigations conducted in many parts of the world result in the same outcome.

X X

SC.912.N.1.6 Describe how scientific inferences are drawn from scientific observations and provide examples from the content being studied.

X X X X

SC.912.N.1.7 Recognize the role of creativity in constructing scientific questions, methods and explanations.

X X X X X

SC.912.N.2.4 Explain that scientific knowledge is both durable and robust and open to change. Scientific knowledge can change because it is often examined and re-examined by new investigations and scientific argumentation. Because of these frequent examinations, scientific knowledge becomes stronger, leading to its durability.

X

SC.912.N.2.5 Describe instances in which scientists' varied backgrounds, talents, interests, and goals influence the inferences and thus the explanations that they make about observations of natural phenomena and describe that competing interpretations (explanations) of scientists are a strength of science as they are a source of new, testable ideas that have the potential to add new evidence to support one or another of the explanations.

X

SC.912.N.4.1 Explain how scientific knowledge and reasoning provide an empirically-based perspective to inform society's decision making.

X

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Benchmark Lesson

1 2 3 4 5 6 7

SC.912.N.4.2 Weigh the merits of alternative strategies for solving a specific societal problem by comparing a number of different costs and benefits, such as human, economic, and environmental.

X

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Background Information

General background information is given here. More information can be found within the individual lessons as well as in the Silkworm Husbandry and Classroom Care information pages. The technology involved with the advent of gene silencing has been increasing at an exponential rate. Although the discovery of this complex method of protein regulation only began in 1998, the advancement of specie genomes and their gene positions has been accomplished at a run-away pace. The discovery began with a most inconspicuous creature, the Caenorhabditis elegans or C. elegans. Discovery of RNA Interference or RNAi During the research of Andrew Fire and Craig Mello on gene expression in the worm Caenorhabditis elegans, they found that injecting mRNA that encodes for muscle protein production elicited no responses from the worms. Bear in mind that the genetic code in the mRNA is considered as the sense sequence. They also tried to inject antisense RNA into the worms which can pair with the sense sequence mRNA but it also elicited no responses from the worms. Finally, when they tried to inject both the sense and the antisense RNA together, they noted twitching movements from the worms. These results surprised them since they know that the same kinds of movements were noted from worms whose genes encoding for muscle protein were dysfunctional. To explain the results that they got, Fire and Mello hypothesized that the double-stranded RNA molecule formed by the binding of the sense and antisense RNA silences the gene carrying exactly the same code as the RNA molecule. To test their hypothesis, they injected double-stranded RNA that codes for specific proteins. In all their experiments, they found that the genes carrying exactly the same code as the RNA they injected were silenced. Their discovery on RNA interference is noteworthy for two reasons. First, with RNAi, researchers can specifically knockdown the production of any protein in a cell. Second, initially scientists thought that a portion of the DNA called introns were just junk DNA and they serve very little purpose, buy now they know that much of these introns code for RNAi elements. Mechanisms of RNA Interference or RNAi Double-stranded RNAs or dsRNA are used to silence the expression of target genes via RNA interference. After introduction of the dsRNA into an organism, it is processed and broken down into small interfering RNA or siRNA by an enzyme called Dicer. Then, siRNA bind to RNA-induced silencing complexes or RISCs. The siRNA strands then guide the RISCs to their complementary RNA where they cleave the cognate RNA leading to a generalized destruction of the RNA and cessation of protein synthesis if mRNA was the target. Clinical Relevance of the Discovery RNAi is an important process in our defense against viruses. A lot of viruses have double-stranded RNA in their genome and when such a virus infects a cell, it injects its RNA molecule which will bind to Dicer which will eventually lead to the degradation of the viral RNA. Other proposed medical uses of RNAi technology is in the field of disease management and treatment. If we can create double-stranded RNA that can degrade the mRNA of genes responsible for chronic diseases like hypertension, diabetes, cancer, cardiovascular diseases and other medical conditions, we can silence these genes and control these diseases. The only drawback of this application of RNAi is its safety. RNAi has a potential for off-target effects in which a gene similar to the target gene can also be silenced. Such potential can cause adverse effects on the health of the patients.

http://en.wikipedia.org/wiki/RNA_interference

http://www.ambion.com/techlib/append/RNAi_mechanism.html

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Silkworm Husbandry FROM EGG TO ADULT - COMPLETE METAMORPHOSIS (EGG, LARVA (5 INSTARS), PUPA, ADULT) General Information: Bombyx mori = the common silkworm is a specimen indigenous to China

About Silkworms

General Information, Caring For & Breeding Silkworms Background

Silkworms are the larva of a moth (Bombyx mori) native to Asia that spins a cocoon of fine, strong, lustrous fiber that is the source of commercial silk. The culture of silkworms is called sericulture. The various species of silkworms raised today are distinguished by the quality of the silk they produce. Silkworms feed on the leaves of the mulberries (genus Morus) and sometimes on the Osage orange (Maclura pomifera). Bombyx Mori will not bite, making an ideal worm for feeding most reptiles, amphibians and other animals, and they offer great nutritional value.

Mulberry Leaves

Newborns are small enough for most baby reptiles to eat and young silkworms can even be fed so they will grow to a desired size. Silkworms are soft-bodied, slow moving and can grow to 3 inches in length. They are also relatively fast growing, reaching about 3 inches in length and ready to cocoon in as little as 25 - 28 days.

Silkworm and Cocoon

Silkworms go through four stages of development, as do most insects: egg, larva, pupa and adult. Click here to see a life cycle chart. The adult (imago) stage is the silkworm moth. The larva is the silkworm caterpillar. The pupa is what the silkworm changes into after spinning its cocoon before emerging as a moth. Since the silkworm grows so much, it must shed its skin four times while it is growing. These stages-within-a-stage are called instars. Today, the silkworm moth lives only in captivity. Silkworms have been domesticated so that they can no longer survive independently in nature,

particularly since they have lost the ability to fly. All wild populations are extinct. Also contributing to their extinction is the extraordinary fact that they only eat mulberry leaves. Silkworms have been used by researchers to study pheromones or sexual attractant substances. The pheromones are released by female moths and the males detect the chemicals with olfactory hairs on their antennae. This allows the male to find the female for mating. The male antennae are made of many small

http://www.silkwormshop.com/images/lifecycle.jpg

http://www.silkwormshop.com/shop.html

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hairs to increase the chances of picking up small amounts of the pheromones over long distances. How to Grow Your Worms to the Perfect Size

Silkworm Eggs and Moth

The great thing about silkworms is that they only grow as much as you feed them, and they can go for up to a week without food. Keep in mind, however, that silkworms become dehydrated after a few days without food, and should be feed at least once daily in order to remain healthy. But, in general, if you have too many silkworms you can feed them a few times per week and they'll stay alive until you need them without growing too much larger.

Wash hands thoroughly before handling the worms or the food or they may develop bacterial problems. Using a cheese grater, grate a small amount of food onto the worms and repeat until the worms reach the desired size. For best results, maintain temperatures between 78° and 88° Excessive condensation forming in the container after feeding is the leading cause of failure. If this condensation does form, take the lid off your container and allow the container and old food to completely dry out. In the future, make sure the previous food is dry before feeding again. Old damp food is a breeding ground for mold and other problems, dry food is not.

Newly hatched silkworms are tiny

As the silkworms grow, you may need to transfer your worms to a larger plastic container. The lid needs to have ventilation holes. If not, you need to vent the lid so the silkworms won't suffocate and to allow condensation to dissipate. You can also use a shoebox. The old food and waste matter can be removed, but does not have to be if it remains thoroughly dry. Under ideal conditions (78° to 88° F and allowed to feed nearly continuously) silkworms can go from egg to 1 inch in length in about 12 days, and 3 inches in under 30 days. The worms will begin to spin cocoons at about 28 - 30 days old or when they are between 2 1/2 and 3 inches long. For more detailed silkworm care instruction, please see our FAQ page. From Cocoon to Moth

Silkworm Moth

Silkworm moths emerge from their cocoons after spending about two to three weeks metamorphosing. As moths, they do not eat or fly. They will usually mate, lay eggs and die within a week. Fertile eggs turn from yellow to gray or purple in a week or so. If the eggs don't hatch within 3 weeks, they usually will not hatch until the following year (see above—from egg to larva).

http://www.silkwormshop.com/faq.html#care

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Proper Handling Procedures Again, in order for your worms to stay healthy for many weeks, you'll need to keep the silkworms as dry as possible. If condensation builds up during feeding, vent the container lid to prevent excess humidity. Be sure to wash your hands thoroughly before handling the worms or their food. Silkworms can be susceptible to bacteria if you don't properly handle them. As long as the container environment remains dry, your worms will be fine. Mold develops from high temperatures and high humidity. If the worms are covered with droppings, silk and old food for too long, mold may develop and kill the worms. If mold does develop, grate about 1/4 inch of food (sold separately) all over the worms with a cheese grater. As the worms crawl to the top of the new food pile you can transfer them off the moldy food and place them into a new container.

GENERAL CLASSROOM SILKWORM CARE:

Silkworm eggs can be purchased from Carolina Biological or other biological supply house.

Silkworm eggs can be purchased from several independent businesses throughout the US.

Eggs can be “hatched” by placing in a petri dish with a substrate of filter paper (cut to size) this will allow you to view the <1mm larva at emergence. (approximate hatch time ≈ 3 – 5 days.

The larva will stay in the petri dish (for the most part) you will NOT need a lid.

Place freshly emerged mulberry leaves (one at a time) into the petri dish (directly on top of the worms)

Replace leaf (withered or venated) as needed. Soon they will be eating multiple leaves

Keep picked leaves in a zip-lock bag with a small amount of water in the refrigerator. (they will keep for many days)

Make sure you find multiple locations for mulberry leaves (employ your students!)

As worms grow allow for larger containers (they will always stay together as long as there is food - and other larva) Container size is minimal as the closer to food and each other the better off they seem to do.

Remove any desecrated larva or leaves. Replace container if evidence of mold appears.

As they feed they also excrete!! When larva get large enough you will be able to gently handle them, remove them to new containers to get rid of leave structures and excrement.

When worms have reached each instar they will often slightly attach themselves with silk to “climb out of their skin” this “anchoring” assists them in the skin removal.

At the fifth instar the larva will pose a “praying” position as they raise their heads up and do not feed. Allowing them to get to this stage will give you good indication that pupation is on the way.

After “praying position” is achieved, place the worms in a large cardboard box, often times the first to pupate will climb corners and spin between the angled sides. When this happens, take remaining larva and put in a single ½ piece of newspaper – rolling like

https://www.silkwormshop.com/shopping/agora.cgi?product=chow

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a big cigar. Fold ends down about an inch and staple ends. Place 2 worms in each rolled paper. You will have a bundle of “cigars” which will go in a cool dark closet stacked at a slight angle.

Approximately 2-3 days after “cigaring” check a couple of “cigars” to see if the larva have cocooned. If so, unwrap papers after a couple of days. Cocoons can be left on desk for students to watch.

After approximately 2-3 weeks, worms will begin to emerge. Keep them in a paper box lid to allow some boundaries. Adult moths neither eat nor fly!! The moths will flap wings vigorously and send pheromones out to attract mates.

Once “bound” with their mates, the moths will “mate” for a few days. Eventually, they will separate and the female will deposit eggs – everywhere. Each adult can lay up to 300 or so eggs! Keep these eggs in a small vial for next hatching. As the egg ages it will darken to indicate fertility. Don’t leave out too long as they will begin to develop. The eggs will keep in the refrigerator for as much as a year.

Adults will shortly die (within 4-7 days upon emergence – likely from dehydration)

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LESSON ONE: Eat – “Sleep” – and Be Quiet! KEY QUESTIONS: What is Gene Silencing? What were/are the dogmatic gene expression pathways? What are the current theories on gene regulation? How do cellular components construct / regulate protein production? Why use Silkworms? KEY SCIENCE TOPICS: Biotechnology protocols/practices, fundamental cellular and molecular mechanisms, animal husbandry, applied genetics, translational research OVERALL TIME ESTIMATES: 110 minutes (60 minutes – prep copies, amino acid game, protein model baggies, 50 minutes – class time) LEARNING STYLES: Visual, auditory, kinesthetic VOCABULARY: Bombyx mori: Scientific name of the most common form of silkworm, proposed to originate in China Amino Acids: An organic molecule containing an amino group (-NH2), a carboxyl (-COOH) group, and a variable side chain (R group) that distinguishes the amino acid. Proteins are synthesized from amino acids. Proteins: A linear polymer built from approximately 20 different amino acid types. The type and sequence of these amino acids are specified by the DNA, this sequence also determines shape and function of the protein. Enzymes: Any of numerous proteins produced in living cells that accelerate or catalyze chemical reactions. Gene Silencing: A method of regulating gene expression using components that offer “missense” such as RNAi and siRNA – any of which interrupts the general formation of the typical protein formation. RNAi’s / siRNA’s: Ribonucleic Acid interference / small interfering Ribonucleic Acid: Genetic sequences “built” to interfere with protein production – often utilizing “missense” or incorrect base pairing. A regulatory mechanism for protein output. tRNA: An abbreviated expression for Transfer Ribonucleic Acid. This genetic sequence is responsible for the “gathering” of amino acids within the cell’s cytoplasm. mRNA: An abbreviated expression for Messenger Ribonucleic Acid. This genetic sequence is responsible for “coding” a gene within the DNA for the instruction of constructing proteins within the cell’s cytoplasm at a ribosome. Polymerase: The enzyme responsible for DNA replication and RNA formations. Ribozymes: An RNA molecule with a tertiary structure that often performs catalytic (enzymatic) functions. It is known to self-cleave which consumes it’s own structure. Ribozyme means: ribonucleic acid enzyme Transcription: The process of RNA production within the nucleus using DNA as a “template” to copy a specific location within the DNA (typically a gene). The DNA is “transcribed” from the 5’3’ using nucleotides. Translation: A “decoding” of the mRNA (created in transcription) at a ribosome within the extra-nuclear space. The process produces primary structure proteins that can be later modified to perform an assortment of structural/physiological functions.

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Complete Metamorphosis: Changes that occur within the life-cycle of many organisms. The terms typically refer to entomological organisms. Complete metamorphosis: egg, larva, pupa, adult. Incomplete Metamorphosis: Changes that occur within the life-cycle of many organisms. The terms typically refer to entomological organisms. Incomplete metamorphosis: egg, nymph, and adult LESSON SUMMARY: The students will be introduced to the concept of gene silencing with a short PowerPoint that highlights cellular and molecular mechanisms, as well as a generalized view of silkworm life cycles. The fundamental understanding of these mechanisms is crucial in the development of the unit. A 20 question pre-investigation assessment will be given to produce a knowledge baseline. The two activities (amino acid bingo and primary protein modeling) are intended to reinforce the concepts as well as produce a heightened interest. STUDENT LEARNING OBJECTIVES: The student will be able to…

1. Characterize the general functions of cellular organelles. 2. Differentiate between the current views on gene expression and protein production. 3. Explain the general process of Transcription and Translation. 4. Compare the stages of complete metamorphosis in Bombyx mori. 5. Generalize the role of RNAi’s in gene silencing. 6. Produce protein models using the codon chart in amino acid selection.

STANDARDS SC.912.L.14.2 SC.912.L.14.3 SC.912.L.14.6 SC.912.L.16.3 SC.912.L.16.4 SC.912.L.16.5 SC.912.L.16.6 SC.912.L.16.7 SC.912.L.16.8 SC.912.L.16.9 SC.912.L.16.10 SC.912.L.16.11 SC.912.L.16.12 SC.912.L.18.1 SC.912.L.18.4 SC.912.L.18.11 SC.912.N.1.1 SC.912.N.1.6 SC.912.N.1.7 MATERIALS:

1 copy of Teacher Pages (when making a copy of Teacher Page – highlight “teacher page bar” and delete to make a master copy): Pre-investigation assessment – Biotechnology, Molecular and Cellular Mechanisms, Gene Silencing (make 25 copies for one/student)

1 copy of Teacher Pages: PowerPoint handout of Transcription/Translation Overview

3 copies of Teacher Pages: 1 class set (25) of Amino Acid Bingo Cards (assortment of 3 master copies)

25 cups of fruit loops (or similar “markers”)

1 copy of Universal Codon Chart (make 25 copies for one / student)

1 copy of Teacher Pages: 25 copies of Primary Protein Modeling Worksheet

25 prepared baggies containing ingredients of amino acid colored beads, mRNA cards, pipe cleaner

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PowerPoint file on “Introduction to Molecular and Cellular Mechanisms and the Life-Cycle of Bombyx mori”

BACKGROUND INFORMATION: Instructors should be fully versed in cellular and molecular fundamentals involving organelle function and specifications. An overview of organelles such as: ribosomes – site of protein synthesis; nucleus – control center of cell, contains genetic information held within the sequences nucleotides found in the DNA (deoxyribonucleic acid); mitochondria – the “powerhouse of the cell”, necessary in conversion of organic energy into chemical energy; lysosomes – “cell digester”, an enzymatic factory that breaks down large cellular components; endoplasmic reticulum – a transport mechanism, also involved in the production of secretory proteins; golgi apparatus – a storage and packaging facility primarily involved with organizing proteins, as well as formation of lysosomes; cytosol – the liquid environment of the cell’s interior (outside the nucleus), contains all organelles and intracellular substances necessary for cell productivity and life. In addition, familiarity with both processes of transcription and translation in relation to gene expression and protein formation will be necessary in review with students. Transcription can be best described as the copying of DNA into smaller “packets” of instructions called RNA. A generalization of the process can be summarized with the following: DNA “unzips” at a particular gene (specific location on a chromosome (chromatin – diffused DNA) that provides instruction for a particular protein or protein component), free floating building blocks of the RNA called nucleotides (made up of a phosphate group, nitrogenous base (A – adenine, G – guanine, C – cytosine, U – uracil), and a ribose sugar) attach to their complimentary base pairs (A with U, and C with G) found on one “side” of the DNA, the bases are bound with an enzymatic “engine”, when complete the RNA (now called mRNA for “messenger”) is released and modified to eventually leave the nucleus to be attached to a ribosome (where the protein will be assembled). A good cue for the students in remembering this process as opposed to Translation is that Transcription is like a “scribe” writing down the DNA code into a language called RNA (one that the ribosome can understand). Translation can best be described as the process of protein formation. Because ribosomes do not “understand” DNA language it must be “translated” to the ribosome in the language called RNA. The process can be summarized as follows: mRNA attaches to a ribosome (made up of a small and a large subunit(s). The mRNA sequence of nucleotides is “read” by the ribosome in triplet bases called codons. An additional RNA called tRNA (for transfer RNA) resides in the cytosol. tRNA has the job description of “gathering” the building blocks of proteins called amino acids (also located in the cytosol). The amino acids are differentiated by the tRNA (because tRNA have triplet base sequences called “anticodons”) and brought to the ribosome to pair anticodon with codon. Amino acids are then “dropped off” and bound to one another (by a peptide bond) in a growing chain that will eventually become the desired protein. This process is also assisted by another RNA called rRNA (for ribosomal RNA). The instructor should be familiar with the life cycle of Bombyx mori (common silkworm). This should include such aspects as: complete metamorphosis and their approximate time line of development, physical characteristics of each metamorphic “stage”, dietary and environmental requirements, and reproductive overview. (Silkworm Husbandry found in preface) The primary purpose of this investigation is to help the students understand the use and future of RNAi and siRNA technologies. The instructor should be well-versed in these methods of protein regulation. In summary - the process of protein regulation using interfering RNA’s is relatively new in the scope of molecular biology’s history. However, much is known and utilized (especially in the identification of gene “purpose” and location) within the research laboratories. We would like to implement the technique in our high school classrooms, in our investigation with silkworms. Essentially, the siRNA targets a specific location on the mRNA. Because it is meant to “mimic” the gene instructions there are purposeful “alterations” made to the siRNA that cause it to differ “slightly” from the original. Cellular “police” then “disengage” targeted gene and any others that even closely resemble the targeted gene. This disengagement disallows the production of the protein and thus gains the desired outcome. ADVANCE PREPARATION:

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1. Prepare all copies of above-mentioned Teacher Pages. 2. Prepare PowerPoint for class viewing. 3. Prepare protein modeling “baggies” with: colored beads (2 each of yellow, blue, green, orange, purple,

and pink) that represent given amino acid colors on handout, 12 inches of pipe cleaner, 10 cards that indicate necessary mRNA codon for amino acid selection, and Universal Codon Chart.

4. Prepare cups of bingo “markers” (i.e. fruit loops or other small items easily obtained). 1 cup/student. 5. Prepare tongue depressors with mRNA codons written on ends for random drawing (64 needed)

PROCEDURE AND DISCUSSION QUESTIONS WITH TIME ESTIMATES:

1. (10 minutes) Administer 25 question pre-investigation assessment for baseline knowledge. Collect. 2. (10 minutes) Introduction of unit study on Gene Silencing and Silkworms with PowerPoint (students

follow along, yet, they have a copy for future reference. This powerPoint sets “the stage” for the investigation by providing students a visual introduction to capture their interest, put a “starter image” in their minds to events that will proceed, and offer a “gradual” beginning to the investigation. In addition, the presentation will highlight the sequence of DNA to RNA to Protein (how they are assembled), and how gene silencing is a method of protein regulation. This PowerPoint will also be useful in tying in the formation of proteins in silk production of silkworms and our desire to demonstrate protein silencing techniques (regulation) using siRNA and RNAi.

3. (15 minutes) Amino Acid Bingo – distribute Amino Acid Bingo Cards, cups of markers, and Universal Codon Chart (found as separate handout) handout. Instructor will randomly pull from tongue depressors, call out codon sequence, students will use protein modeling sheet to select appropriate amino acid (3-letter identification of amino acid is standard protocol), student then finds 3-letter amino acid on playing card if present (placing marker). All cards have a “free” middle marker. When student completes a row/column/or all four corners they must shout out “AMINO”. Instructor may reward with candy or some other prize. If time allows, have students clear their cards and repeat. When finished, collect all cards and markers (do not eat the markers!)

4. (10 minutes/remaining time) Primary Protein Modeling – instructor distributes “baggies” of ingredients, instruct students to write down which group of cards they have (located on each card) on handout and proceed with each card to identify appropriate codon to amino acid (and subsequent bead). Beads are then “woven” onto the pipe cleaner until complete. Students write down proper amino acid colors in provided blanks, and then answer 5 questions on handout. When complete, replace components for next session. Collect handouts.

5. (5 minutes) Review and discuss objectives, share with students DAY 2 plan. ASSESSMENT SUGGESTIONS:

Collection and grading of pretest should give instructor a generalized idea of student and class base knowledge. (keep tests for future comparison and return)

Student worksheet on Primary Protein Modeling can be checked for completion and understanding (return).

EXTENSIONS: ACTIVITIES:

Demonstrate a comparison/contrast of complete and incomplete metamorphosis to demonstrate other life cycles (use beetles, flies, dragonflies, grasshoppers, bees etc…)

View projected cells as a class to help in the understanding of cellular components

Demonstrate the fragility of proteins to pH and temperature changes (egg whites, cooking with acids) REFERENCES: Next Generation Sunshine State Standards: www.fldoe.org/bii/curriculum/sss/ Vocabulary: www.biology-online.org, www.freedictionary.com, http://en.wikipedia.org

http://www.fldoe.org/bii/curriculum/sss/

http://www.biology-online.org/

http://www.freedictionary.com/

http://en.wikipedia.org/

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Teacher Pages: Primary Protein Modeling – Teacher Answers

mRNA (Messenger RNA) Genetic “Codon” Code for Amino Acids

First Letter Second Letter

U C A G

U Phenylalinine (UUU) Serine (UCU) Tyrosine (UAU) Cysteine (UGU) U

Phenylalinine (UUC) Serine (UCC) Tyrosine (UAC) Cysteine (UGC) C

Leucine (UUA) Serine (UCA) Stop (UAA) Stop (UGA) A

Leucine (UUG) Serine (UCG) Stop (UAG) Tryptophan (UGG) G

C Leucine (CUU) Proline (CCU) Histidine (CAU) Arginine (CGU) U

Leucine (CUC) Proline (CCC) Histidine (CAC) Arginine (CGC) C

Leucine (CUA) Proline (CCA) Glutamine (CAA) Arginine (CGA) A

Leucine (CUG) Proline (CCG) Glutamine (CAG) Arginine (CGG) G

A Isoleucine (AUU) Threonine (ACU) Asparagine (AAU) Serine (AGU) U

Isoleucine (AUC) Threonine (ACC) Asparagine (AAC) Serine (AGC) C

Isoleucine (AUA) Threonine (ACA) Lysine (AAA) Arginine (AGA) A

Methionine / Start (AUG) Threonine (ACG) Lysine (AAG) Arginine (AGG) G

G Valine (GUU) Alanine (GCU) Aspartate (GAU) Glycine (GGU) U

Valine (GUC) Alanine (GCC) Aspartate (GAC) Glycine (GGC) C

Valine (GUA) Alanine (GCA) Glutamate (GAA) Glycine (GGA) A

Valine (GUG) Alanine (GCG) Glutamate (GAG) Glycine (GGG) G

Methionine = purple Arginine = pink Threonine = white

Proline = blue Tyrosine = yellow Glycine = black

Alanine = green Isoleucine = orange Leucine = brown

Instructions: Remove “codons” (cards 1-10) from the baggie. Remove pipe cleaner. Find the correct amino acid from the chart that matches the “codon”. After locating the correct amino acid, find the name in the chart and determine which color bead to string onto the pipe cleaner. All proteins start with “AUG” (purple). Write down the colors below: Group __A__ = purple, ______, ______, ______, ______, ______, ______, ______, ______, STOP 1 2 3 4 5 6 7 8 9 Complete:

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1. Where does TRANSLATION take place specifically? in the cytoplasm within a ribosome or set

of ribosome chains (polysomes)

2. What RNA type gathers the amino acids? mRNA or rRNA or tRNA (circle) 3. What is the chain of amino acids called when complete? primary protein (polypeptide) 4. What is the name of the RNA type that brings the DNA code out of the nucleus? mRNA 5. How many different types of amino acids are there? 20

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Student Pages: Primary Protein Modeling – Student Worksheet

mRNA (Messenger RNA) Genetic “Codon” Code for Amino Acids


U C A G

















Methionine = purple Arginine = pink Threonine = white

Proline = blue Tyrosine = yellow Glycine = black

Alanine = green Isoleucine = orange Leucine = brown

Instructions: Remove “codons” (cards 1-10) from the baggie. Remove pipe cleaner. Find the correct amino acid from the chart that matches the “codon”. After locating the correct amino acid, find the name in the chart and determine which color bead to string onto the pipe cleaner. All proteins start with “AUG” (purple) Write down the colors below: Group _____= ______, ______, ______, ______, ______, ______, ______, ______, ______, STOP 1 2 3 4 5 6 7 8 9 Complete:

1. Where does TRANSLATION take place specifically? ____________________

2. What RNA type gathers the amino acids? mRNA or rRNA or tRNA (circle)

3. What is the chain of amino acids called when complete? _________________

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4. What is the name of the RNA type that brings the DNA code out of the nucleus? ___RNA

5. How many different types of amino acids are there? _____

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Teacher Pages: Amino Acid Bingo: “Scoring Card” – Form A

AMINO ACID “BINGO” (“AMINO”)

STOP

GLY

STOP

ISO

SER

STOP

GLUTA MATE

MET

THR

ARG

CYS

TYR

FREE

START

TRY

ASP

PRO

LYS

GLU

HIS

PHE

VAL

ASPAR TATE

LEU

ALA

ANY 5 IN A ROW IS A WINNER! WHEN COMPLETE, SHOUT OUT “AMINO”!!

FREE IS …. WELL, FREE! PUT A MARKER ON FREE AT THE START OF THE GAME OH – BY THE WAY…FREE AND ALL 4 CORNERS IS ALSO A WINNER!!!

A

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Teacher Pages: Amino Acid Bingo: “Scoring Card” – Form B


LEU

GLY

ASP

SER

TRY

ASPAR TATE

MET

STOP

THR

ARG

ISO

PHE

FREE

GLU

LYS

STOP

START

CYS

ALA

HIS

GLUT MATE

VAL

STOP

TYR

PRO



B

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Teacher Pages: Amino Acid Bingo: “Scoring Card” – Form C


TYR

GLY

ASP

CYS

LYS

ASPAR TATE

GLUTA MATE

PHE

THR

ARG

ISO

STOP

FREE

ALA

TRY

STOP

PRO

SER

GLU

HIS

MET

VAL

STOP

LEU

START



C

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Teacher Pages: Amino Acid Bingo: Instructor Directions

Prepare “copies” of scoring cards (roughly 1/3 of class for each)

Prepare ‘drawing sticks” (use tongue depressors – write mRNA “codon” on the ends)

Prepare markers (use small Dixie cups (or equivalent) place approximately 25 “markers” (Fruit Loops work great!) in each.

Randomly distribute scoring cards

Distribute markers (make sure they are not eaten – you need to use them for the next class!)

Give instructions: o Say: “I will randomly draw from depressors and read of the codon – you look up the sequence on

the protein modeling page – locate the correct amino acid and see if it is found on your scoring card, if so, place a marker on the square, when you have five in a row (potentially counting the free) or get all four corners – shout out “Amino” or are not “first” you don’t win!”

o Say: “Oh, by the way codons, will only be repeated one time!” o Say: “Please don’t eat the markers – many others already have touched them!”

Check accuracy of winner by having them read back the amino acids o Check with “pulled” drawing sticks

Repeat game by returning “drawing sticks” and begin with “clean” scoring cards (remember to place a marker on Free!)

QUICK GUIDE TO CODONS AND RELATED AMINO ACIDS:


U C A G

















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Teacher Pages: Introduction Powerpoint – Silkworm Life-Cycle - Agenda

“Silence of the Genes”

A Bio-Tech Study in Gene Silencing

and RNA(interference)

Silkworm - Life Cycle - egg

Silkworm - Life Cycle - larvae (kegos)

Silkworm - Life Cycle - larvae 5th instar

Silkworm - Life Cycle - cocoon/pupae

Silkworm - Life Cycle - adult hatch

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Silkworm - Life Cycle - adult

Abstract

Gene silencing is arguably the future of modern personal medicine. Although

much work has been conducted on non-human candidates the advancement

shows determined promise. This study involves the use of silkworms and an

attempt to silence the silk producing gene (to quantitative amounts). Although an

industrial conflict in practice, the procedure will: demonstrate technique, bolster

future RNAi study, potentially lead to an enhancement in silk cocoon production

(greater market values), and be very cool. The silkworms will be raised on their

preferred diet of mulberry leaves (chow) through their larval development.

Throughout their instar molts, varying groups will be given chow enhanced with

manufactured ingestible RNAi. Controls will include non-influenced larva, as well

as larva fed with ingestible dyes (to demonstrate silk enhancements through

ingestible variables). Quantitative results will be determined by visual and silk

mass assessments.

Agenda

DAY 1 - Introduction, Pre-Test assessment, overview of Cellular/Molecular mechanisms,

Amino Acid Bingo, Protein Primary Chain Construction

DAY 2 - Jigsaw (an investigation of silkworm history/economics/life-cycle)

Student collaboration on reverse genetics and gene silencing

DAY 3 - Corneal Repair and RNAi clinical uses, Video - RNAi and Gene Silencing,

Review bio-tech practices (i.e. gel-electrophoresis, pipetting, DNA extraction)

DAY 4 - DNA extraction of silkworm metamorphic stages, PCR (silk gene amplification)

DAY 5 - RNAi selection techniques - web-based investigation, gel-electrophoresis of

amplified silk gene (with membrane gene), prep leaves with ingestible RNAi

DAY 6 - Student Research (clinical applications of gene silencing (projected/ongoing)),

production of student powerpoint (for presentation)

DAY 7 - Student PPT Presentations, wrap-up discussions

DAY 8 - Student PPT Presentations, wrap-up discussions (if necessary)

FINAL DAY - Wrap-up discussions, data collection/comparisons, post-test assessment

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Teacher Pages: Universal Codon Chart


U C A G

















36

Teacher Pages: Example of protein modeling card (1 of 10)

8 C

UAA

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LESSON TWO: Who Put the “i” in my RNA? KEY QUESTIONS: What is RNA interference? How does RNAi work? What are the applications of RNA interference?

OVERALL TIME ESTIMATES: 50 minutes (Part 1: 15 minutes, Part 2: 20 minutes, Part 3: 15 minutes) LEARNING STYLES: Visual and auditory VOCABULARY: Argonaute: Protein that is the main component of the RNA induced silencing complex (RISC) and aids in the recognition and cleavage of mRNA during RNAi. Dicer: A nuclease that cleaves double-stranded RNAs. Dicer processes long dsRNA into siRNA or miRNA or 21-23 nucleotides. miRNA: Micro-RNAs (miRNAs) are single-stranded RNAs of 21-23 nt. Similar to siRNAs, miRNAs can silence gene activity via destruction of homologous mRNA. RISC: The RNA induced silencing complex (RISC) is composed of siRNA (or miRNA), Argonaute protein, and other proteins that catalyzecleavage of the mRNA target. RNAi: RNA interference (RNAi) is the process of using small pieces of double-stranded RNA to reduce the activity of specific genes. The process occurs naturally in many organisms and is nowcommonly used in basic research. siRNA: Small interfering RNA (siRNA) is 21-23 nt double-stranded RNA molecules formed from the cleavage of long dsRNA by dicer. The siRNA guides the cleavage and degradation of its targetmRNA. LESSON SUMMARY: Using a jigsaw approach, students will learn about the fundamentals of RNA interference. In Part 1, students are divided into 6 groups: Argonautes, C. elegans, Dicers, RISC, RNAs, and siRNAs. Each group is given one or two resources and a set of questions for each resource. When they have completed Part 1, they will then switch into of four groups for Part 2. Part 2 has a student worksheet with more general questions about RNAi. As a group, the students need to complete the Part 2 worksheet. For Part 3, students will return to their original group and draw a diagram of the process of RNA interference. They will work out their ideas on white boards, and then transfer their final diagram to the Part 3 worksheet. STUDENT LEARNING OBJECTIVES: The student will be able to…

1. Describe the mechanism and components involved in RNA interference. 2. Compare and contrast siRNA and miRNA. 3. Describe the uses of gene silencing in basic research, agriculture, and disease research and treatment.

STANDARDS: SC.912.L.14.6 SC.912.L.16.3 SC.912.L.16.4 SC.912.L.16.5 SC.912.L.16.7 SC.912.L.16.10

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MATERIALS:

Part 1. o Per Group: Computers with internet access for 3 groups (Argonautes, Dicers, RISC).

Copies of the articles assigned to their group. Optional: Headphones for viewing the online videos and interactives.

o Per Student: Part 1 Worksheet.*

Part 2. o Per Student: Part 2 Worksheet.*

Part 3. o Per Group: White board and markers

Part 3 Worksheet* Colored pencils/markers

*To reduce confusion among the three worksheets, copy them on different colored paper. BACKGROUND INFORMATION: Instructors should review the resources students will use in this lesson. Be familiar with the vocabulary and the process of RNA interference. ADVANCE PREPARATION:

1. Make copies of student worksheets. 2. Make copies of the articles used as resources. 3. Arrange the classroom into six stations. Three of the stations need computers with internet access. 4. Put white boards and markers at each station (needed for Part 3). 5. Put colored pencils or markers at each station (needed for Part 3).

PROCEDURE AND DISCUSSION QUESTIONS WITH TIME ESTIMATES: Part 1 (15 minutes)

1. Divide the students into the six expert groups (Argonautes, C. elegans, Dicer, RISC, RNAi, siRNA). 2. Assign each person in the group a number: 1, 2, 3, or 4 (to group them for Part 2) 3. Pass out the resources and worksheets for each group. 4. Be sure the Argonautes, Dicers, and RISC have computers (and optional headphones) 5. Instruct students to complete the Part 1 worksheet as a group. Make sure that everyone understands the

concepts contained in their resources-they are going to have to explain it to other students in Part 2. Part 2 (20 minutes)

1. Have students move to their second group (1, 2, 3, or 4). Each group should have one Argonaute, one C. elegan, one Dicer, one RISC, one RNAi, and one siRNA.

2. Pass out the Part 2 worksheet. 3. Each student will have read a different article or watched a different video clip or animation. Students

will need to discuss what they learned in their first groups to complete the Part 2 worksheet. While some of the questions from Part 1 are duplicated on the Part 2 worksheet, the Part 2 worksheet asks more comprehensive questions which require the students to collectively synthesize the information from Part 1.

Part 3 (15 minutes) 1. Put students in their expert groups (Argonautes, C. elegans, Dicer, RISC, RNAi, siRNA). 2. Tell students that they are now going to diagram the process of RNA interference. Point out the

terms/molecules they need to include in their diagrams. 3. Give each group their supplies (whiteboards or chart paper, markers, Part 3 worksheet, colored

pencils/markers) 4. Instruct students to brainstorm ideas on the whiteboards and then when they are satisfied transfer their

diagrams to the group worksheet.

39

ASSESSMENT SUGGESTIONS:

Check student worksheets and diagrams for completion and/or accuracy. EXTENSIONS:

Students research the uses of RNAi and make a poster/presentation to the class. REFERENCES: Vocabulary: http://www.rnaiweb.com/RNAi/RNAi_Glossary/ An Introduction to Reverse Genetic Tools for Investigating Gene Function http://www.apsnet.org/edcenter/advanced/topics/Pages/ReverseGeneticTools.aspx Discovery of RNA inference http://www.experiment-resources.com/rna-interference.html Explained: RNA interference http://web.mit.edu/newsoffice/2009/explained-rna.html Gene Silencing RNA Interference (RNAi): a new pioneering discovery http://www.gmo-safety.eu/basic-info/466.rna-interference-rnai-pioneering-discovery.html Gene-silencing drug can halt and reverse deadly brain disorder http://www.scotsman.com/the-scotsman/health/gene-silencing-drug-can-halt-and-reverse-deadly-brain-disorder-1-2366311 Learn More About RNAi http://www.nature.com/nrg/multimedia/rnai/animation/index.html NOVA: RNAi Expert Q & A http://www.pbs.org/wgbh/nova/body/hannon-rnai.html NOVA: RNAi Explained http://www.pbs.org/wgbh/nova/body/rnai-explained.html NOVA: The RNAi Cure http://www.pbs.org/wgbh/nova/body/rnai-explained.htm RNA Interference http://www.youtube.com/watch?v=cK-OGB1_ELE http://www.nature.com/nrg/multimedia/rnai/animation/index.html

http://www.rnaiweb.com/RNAi/RNAi_Glossary/

http://www.apsnet.org/edcenter/advanced/topics/Pages/ReverseGeneticTools.aspx

http://www.experiment-resources.com/rna-interference.html

http://web.mit.edu/newsoffice/2009/explained-rna.html

http://www.gmo-safety.eu/basic-info/466.rna-interference-rnai-pioneering-discovery.html

http://www.scotsman.com/the-scotsman/health/gene-silencing-drug-can-halt-and-reverse-deadly-brain-disorder-1-2366311


http://www.nature.com/nrg/multimedia/rnai/animation/index.html

http://www.pbs.org/wgbh/nova/body/hannon-rnai.html

http://www.pbs.org/wgbh/nova/body/rnai-explained.html

http://www.pbs.org/wgbh/nova/body/rnai-explained.htm

http://www.youtube.com/watch?v=cK-OGB1_ELE


40

Teacher Pages: Jigsaw Activity Group Assignments

Group Assigned Resource Computer/Internet

Access needed?

Argonautes Learn More About RNAi (Interactive) http://www.nature.com/nrg/multimedia/rnai/animation/index.html

Yes

C. elegans Discovery of RNA Interference http://www.experiment-resources.com/rna-interference.html NOVA_RNAiExpert Q & A http://www.pbs.org/wgbh/nova/body/hannon-rnai.html

No

No

Dicer NOVA_RNAi Explained (Interactive) http://www.pbs.org/wgbh/nova/body/rnai-explained.html Explained: RNA Interference http://web.mit.edu/newsoffice/2009/explained-rna.html

Yes

No

RISC RNA Interference (Nature Reviews Genetics 5 min video) Video on YouTube http://www.youtube.com/watch?v=cK-OGB1_ELE Video on Nature.com http://www.nature.com/nrg/multimedia/rnai/animation/index.html

Yes

RNAi An Introduction to Reverse Genetic Tools http://www.apsnet.org/edcenter/advanced/topics/Pages/ReverseGeneticTools.aspx Gene Silencing RNA Interference (RNAi): a new pioneering discovery http://www.gmo-safety.eu/basic-info/466.rna-interference-rnai-pioneering-discovery.html

No

No

siRNA NOVA_The RNAi Cure http://www.pbs.org/wgbh/nova/body/rnai-explained.html Gene-silencing drug can halt and reverse deadly brain disorder http://www.scotsman.com/the-scotsman/health/gene-silencing-drug-can-halt-and-reverse-deadly-brain-disorder-1-2366311

No

No





http://www.pbs.org/wgbh/nova/body/hannon-rnai.html


http://web.mit.edu/newsoffice/2009/explained-rna.html












41

Silence of the Genes Lesson #2: Who Put the “i” in my RNA? Part 1

Name ____Key_______________________ Period________ Date__________________ Group 1: Argonautes

Directions. Watch the interactive slide show, Learn More About RNAi, and answer the following questions. http://www.nature.com/nrg/multimedia/rnai/animation/index.html 1. What does it mean to “silence” a gene?

Silencing a gene happens when a protein from a specific gene cannot be expressed. 2. What enzyme is responsible for the transcription of DNA into primary mRNA?

RNA polymerase II 3. What is the purpose of the ribosomes in the cytoplasm?

Carries out translation to turn mRNA into proteins (polypeptide chains) 4. What must RNAi target to silence genes?

mRNA 5. What is the difference between exogenous and endogenous siRNA?

Endogenous siRNA is produced in the nucleus and then processed into siRNAs. Exogenous siRNA is produced outside the cell nucleus; they are triggered by the delivery of long-stranded RNA intentionally by scientists or as part of their immune response against viral RNA

6. What is a major use of siRNA in the research laboratory?

to explore the function of genes 7. What is the function of dicer?

cuts RNA into 21-23 nucleotide siRNA or miRNA 8. What is the RISC complex? What does it do?

RISC is the RNA-inducing silencing complex and is composed of the small RNA (siRNA or miRNA), Argonaute protein, and other proteins. It cleaves (cuts) the target mRNA.

9. What part of the miRNA must match the mRNA? Why is this important?

Only the “seed” portion of the miRNA needs to complement the mRNA. This allows each type of microRNA can target many mRNAs.


42

Silence of the Genes Lesson #2: Who Put the “i” in my RNA?

Name ____Key_______________________ Period________ Date__________________ Group 2: C. elegans

Directions. Read the two articles and answer the following questions. Discovery of RNA Interference 1. Who discovered RNAi?

Andrew Fire and Craig Mello (won the Nobel prize in 2006) Both studied Caenorhabditielegans

2. How was RNAi discovered?

Fire and Mello injected dsRNA (double-stranded RNA) into C. elegans that codes for specific proteins and discovered that those genes were “silenced”

3. Why is the discovery of RNAi important (give 2 reasons)

Theoretically, scientists can knockdown or silence any gene and prevent the expression of undesirable proteins. Scientists discovered that introns (which were thought to be “junk” DNA) actually code for RNAi elements.

4. What are some proposed medical uses of RNAi?

Fight viral infections (especially dsRNA viruses) Silence disease causing genes (genes that cause cancer, diabetes, cardiovascular disease, etc.)

5. What are some of the drawbacks of using RNAi?

It might silence the “good” genes along with the “bad” (off-target effects) NOVA_RNAiExpert Q & A 1. Who is the expert in this interview?

Greg Hannon, a molecular biologist on the forefront of the field of RNA interference 2. How do plant viruses protect themselves against RNAi’s?

Viruses have proteins that shut off the plant’s RNAi response. 3. Does RNAi therapy target the patient’s genes or the viral genes?

It could do either. But since viruses are able to mutate or evolve quickly, it might work better to target the patient DNA so that the virus can’t cause an infection.

4. Does RNAi change the genetic sequence?

No, it only affects the expression of a gene. 5. Currently, what is the biggest obstacle to using RNAi technology to treat disease?

The ability to deliver the molecules that trigger RNAi to the right cells in the body

43


Name ____Key_______________________ Period________ Date__________________ Group 3: Dicers

Directions. Watch the RNAi Explained Interactive Slide Show and read the article Explained: RNA Interference. Answer the questions associated with each resource. RNAi Explained (http://www.pbs.org/wgbh/nova/body/rnai-explained.html) 1. How does a cell recognize viral RNA?

The cell recognizes the double-stranded RNA as an invader and mounts a defense against it. 2. What is RNAi? What is its purpose in the cell?

RNA interference It is found naturally in the cell and its purpose is to prevent the replication of viral DNA.

3. What is dicer?

An enzyme that cuts (dices) double-stranded RNA that produces siRNA and miRNA 4. What is RISC?

RNA-Inducing Silencing Complex that unwinds the double-stranded RNA, leaving only one strand; the cut RNA is very unstable and quickly breaks down

5. How does the RISC/RNA complex prevent protein production?

The RISC/RNA complex (which is carrying a single-strand of RNA) finds matching RNA binds to-prevents the production of the viral protein

Explained: RNA Interference 1. What is siRNA?

Short interfering RNA (double stranded RNA molecules) siRNAsbind to complementary mRNA, and along with the RISC complex, prevent the production of a protein (normally coded for by that mRNA)

2. Compare/contrast siRNA and microRNA.

Both siRNA and miRNA are about 21-23 nucleotides in length. Both are involved in gene silencing siRNA is more genotypically specific, microRNA has a more general function


44


Name ____Key_______________________ Period________ Date__________________ Group 4: RISC

Directions. Watch the video, RNA Interference, and answer the following questions. You can find the video YouTube (http://www.youtube.com/watch?v=cK-OGB1_ELE) or Nature Reviews Genetics(http://www.nature.com/nrg/multimedia/rnai/animation/index.html) 1. What is RNAi?

RNA interference 2. What is siRNA? How is it delivered into the cell?

siRNA is small interfering RNA and is either produced by the cell itself or introduced experimentally

3. What endonuclease cuts siRNA and microRNA into smaller segments?

Dicer 4. How large are most siRNAs and microRNAs?

approximately 20 nucleotides long 5. What happens to the siRNA when it binds to the Argonaute protein?

siRNA gets unwound into 2 single strands-the passenger strand, which will be degraded and the guide strand which is incorporated in the RISC complex (RNA-induced silencing complex)

6. The combination of siRNA, Argonaute protein, and other proteins make up what structure?

The RNA-induced silencing complex (RISC) 7. What is the function of the RISC complex?

The RISC complex attaches to specific mRNA (perfect match with siRNA) and cleaves the mRNA with is then degraded.

8. What part of the microRNA pairs with the target mRNA?

the “seed” 10. Which type of small RNA-siRNA or microRNA- can pair with more types of mRNA? Why?

Because the seed microRNA in the RISC complex doesn’t have to complement the mRNA exactly, it can target a much larger number of mRNA. siRNAs have to exactly complement the target mRNA.

11. Argonaute and their small RNA cofactors are found in what type of organisms?

plants, animals, fungi, and some bacteria



45


Name ____Key_______________________ Period________ Date__________________ Group 5: RNAi

Directions. Read the two papers and answer the following questions. An Introduction to Reverse Genetic Tools 1. What is difference between forward and reverse genetics?

In forward genetics, scientists determine the genotype based on the phenotype. They will induce variation/mutation (or observe natural variation) and then develop the genotype based on those variations. Reverse genetics is the opposite process, in which the genetic sequence is known, but the exact function is uncertain. In reverse genetics, scientists change a specific gene or gene sequence and then observed the resultant phenotype.

Forward genetics: phenotype genotype

Reverse genetics: known genotypechange genotype observe phenotype infer gene function

2. What is the goal of reverse genetics? The goal of reverse genetics is to investigate the impact of induced variation and then infer gene function.

3. In what model organism was RNAi first discovered? C. elegans

Gene Silencing RNA Interference (RNAi): a new pioneering discovery 1. What is RNA interference?

Biological mechanism for silencing genes

2. What is siRNA and what is its function? siRNAs are small interfering RNAs that guide the cleavage and degradation of target mRNA

3. How many base pairs is siRNA? 21-23 bp

4. What is the RISC complex? The RISC complex (RNAi induced silencing complex) is composed of an enzyme and the siRNA. RISC cuts the unwanted mRNA so it can’t make the undesired protein

4. At the end of the article, the author gives examples of how gene silencing can be used in plant research and development. Describe the one you find the most interesting.

FlavrSavr tomato (first food GMO)-antisense technique used to delay ripening

RNAi is used to studies genes important in growth behavior and stress resistance

antisense techniques used to reduce the types of starch produced by potatoes

silencing pollen development genes to prevent the unintended propagation of genetically modified plants

46


Name ____Key_______________________ Period________ Date__________________ Group 6: siRNA

Directions. Read the two papers and answer the following questions. The RNAi Cure (NOVA) 1. What are the some of the obstacles to using RNAi technology to treat disease?

how to deliver RNAi drugs to the correct target cells how to prevent shutting down good genes or cellular processes how to make sure the drugs stay active long enough

2. Describe how RNAi might be used to treat the following diseases: Macular degeneration

first use of RNAi technology to treat human disease

RNAi drugs were injected directly into the eye

Patients with macular degeneration product too much VEGF protein which causes an overabundance of blood vessels behind the retina. RNAi drugs shut down the genes that make the protein VEGF.

promising results (25% had significantly better vision; all patients vision had at least stabilized, if it hadn’t improved)

Hepatitis C

RNAi therapy controlled the hepatitis C virus in mice (1st time RNAi therapy had worked in an animal, not just cell cultures)

researchers injected naked RNAi into the tails of the mice, but that isn’t feasible in people.

Scientists are looking at using viral vectors to treat people.

Huntington’s disease

Researchers successfully silenced the Huntington’s gene in mice using RNAi therapy

Unfortunately, they also shut down the healthy version of the gene (patients carry both)

illustrates one of the challenges of using RNAi technology-“off target” effects

HIV

The challenge with using RNAi therapy to treat HIV is that HIV mutates and evolves resistance so rapidly that any single target for an RNAi therapy won’t work

Scientists are looking at a multi-prong attack aimed at several HIV genes. They are also using a couple other therapies (RNA decoys and ribozymes)

Another promising treatment (has worked in mice and rhesus monkeys) is to genetically alter the patient’s bone marrow stem cells with RNAi therapy and then transfuse them

47

back into the patient. These cells would then develop into immune-system cells that fight HIV.

Respiratory Infections

Relatively simple to deliver RNAi drugs directly to the lungs through inhalation.

A very common virus, respiratory syncytial virus (RSV), is a very common virus that infects almost all children by the age of two. It causes cold-like symptoms, but can lead to more serious illnesses like croup, pneumonia, and respiratory failure. It can also be very dangerous to the elderly and people with compromised immune systems.

Scientists have successfully controlled the RSV in mice when they inhale the naked RNA. Cancer

Researchers have silenced several cancer-causing genes with RNAi in cell cultures.

Because of the difficulties in using RNAi therapy to cure cancer, scientists are looking at ways to use RNAi to support chemotherapy. One of the major problems with chemotherapy is that patients develop a resistance to the drugs. A protein called P-glycoprotein destroys the chemotherapy drugs. Scientists have successfully silences the gene that makes this protein in patients suffering from leukemia.

RNAi therapy is also used to study the genes involved in cancer. Gene-silencing drug can halt and reverse deadly brain disorder (Editorial) 1. What is Huntington’s disease?

A genetic disease caused by a toxic protein which leads to uncontrolled movements, loss of mental function, personality changes, and ultimately death.

2. How is RNAi being used to treat Huntington’s disease?

A single dose of RNAi (antisense RNA) blocked out the ability of the Huntington gene to produce the protein that causes the symptoms of Huntington’s disease in mice and rhesus monkeys. The benefits lasted for nine months after the treatment. Scientists hope the treatment will lead to a “huntingtin holiday” (huntingtin is the gene responsible for Huntington’s disease and the protein it produces)

48


Name ____Key_______________________ Period________ Date__________________ Group ______________________________

Part 2 Directions. You will now be placed into another group (Group 1, 2, 3, or 4). Each group will contain a member of each of the original groups (Argonautes, C. elegans, Dicers, RISC, RNAis, siRNAs). You will need to synthesize the information from each member in your group to answer the following questions.

1. What is RNAi? RNA interference. It is used to silence gene expression.

2. Who discovered RNAi? Andrew Fire and Craig Mello (won the Nobel prize in 2006)

3. How was RNAi discovered? Fire and Mello injected dsRNA (double-stranded RNA) into C. elegans that code for specific proteins and discovered that those genes were “silenced”

4. In what model organism was RNAi first discovered? C. elegans

5. What does it mean to “silence” a gene? Silencing a gene happens when a protein from a specific gene cannot be expressed.

6. What must RNAi target to silence a gene? mRNA

7. Describe 3 uses of RNAi technology. Fight viral infections (especially dsRNA viruses) Silence disease causing genes (genes that cause cancer, diabetes, cardiovascular disease, etc.) to explore the function of genes

8. What are FlavrSavr tomatoes? How was gene silencing used to produce these tomatoes? FlavrSavr tomatoes were the first approved food GMO and used antisense techniques to delay ripening

9. What was the first human disease treated with RNAi? macular degeneration

10. How is RNAi being used to treat Huntington’s disease?

A single dose of RNAi (antisense RNA) blocked out the ability of the Huntington gene to produce the protein that causes the symptoms of Huntington’s disease in mice and rhesus monkeys. The benefits lasted for nine months after the treatment.

49

11. How is RNAi being used to treat cancer?

Because of the difficulties in using RNAi therapy to cure cancer, scientists are looking at ways to use RNAi to support chemotherapy. One of the major problems with chemotherapy is that patients develop a resistance to the drugs. A protein called P-glycoprotein destroys the chemotherapy drugs. Scientists have successfully silences the gene that makes this protein in patients suffering from leukemia.

12. What is the difference between forward and reverse genetics? In forward genetics, scientists determine the genotype based on the phenotype. They will induce variation/mutation (or observe natural variation) and then develop the genotype based on those variations. Reverse genetics is the opposite process, in which the genetic sequence is known, but the exact function is uncertain. In reverse genetics, scientists change a specific gene or gene sequence and then observed the resultant phenotype.

13. What is meant by “off-target” effects of RNA interference? Off-target means that the RNAi silences some genes that we don’t want silenced.

14. What enzyme is responsible for the transcription of DNA into primary mRNA? RNA polymerase II

15. What is dicer? What is its purpose? An enzyme that cuts (dices) double-stranded RNA into shorter siRNA and miRNA.

16. siRNA and miRNA are called “small RNAs”. How small are they (how many nucleotides)? 21-23 nucleotides

17. What is the RISC complex? What molecules comprise the RISC complex? What is its function? RISC is the RNA-inducing silencing complex and is composed of the small RNA (siRNA or miRNA), Argonaute protein, and other proteins. It cleaves (cuts) the target mRNA.

18. What happens to the siRNA when it binds to the Argonaute protein? The siRNA gets unwound into 2 single strands-the passenger strand, which will be degraded and the guide strand which is incorporated in the RISC complex (RNA-induced silencing complex)

3. 19. What part of the siRNA must match the target mRNA? What part of the miRNA must match the target mRNA? The complete siRNA must match (complement) the targer mRNA. Only the “seed” part of the miRNA needs to match the mRNA.

19. Which type of small RNA (siRNA or microRNA) can pair with more types of mRNA? Why? Because the seed microRNA in the RISC complex doesn’t have to complement the mRNA exactly, it can target a much larger number of mRNA. siRNAs have to exactly complement the target mRNA.

50

20. Argonaute and their small RNA cofactors are found in what type of organisms? plants, animals, fungi, and some bacteria

21. Does RNAi change the genetic code of a gene? Explain. No, it only affects the expression of a gene by silencing it. The specific genotype still exists.

22. What are some problems associated with using RNAi to treat disease? How to deliver RNAi drugs to the correct target cells How to prevent RNAi from shutting down good genes or cellular processes (off-target effects) How to make sure the drugs stay active long enough to actually silence the gene.

51


Name ____Key_______________________ Period________ Date__________________ Group ______________________________

Directions. Return to your expert group (Argonautes, C. elegans, Dicers, RISC, RNAi’s, siRNAs ). On your whiteboard, draw a diagram that illustrates the mechanism of RNA interference. At a minimum, be sure to include the following in your diagram: long dsRNA, dicer, siRNA, miRNA, Argonaute, RISC, mRNA. Once you are happy with the diagram on your whiteboard, copy it on this paper.

http://www.rnaiweb.com/RNAi/What_is_RNAi/

http://www.rnaiweb.com/RNAi/What_is_RNAi/

52

Student Pages: Jigsaw Activity Group Assignments Silence of the Genes Lesson #2: Who Put the “i” in my RNA? Part 1

Name ______________________________ Period________ Date__________________ Group: ______________________________

Directions. You will be placed in one of 6 groups (Argonautes, C. elegans, Dicer, RISC, RNAi, siRNA). Each group has one or more resources that you need to use to answer a set of questions.

Group Assigned Resource Computer/Inter

net Access needed?

Argonautes Learn More About RNAi (Interactive) http://www.nature.com/nrg/multimedia/rnai/animation/index.html

Yes

C. elegans Discovery of RNA Interference NOVA_RNAi Expert Q & A

No

No

Dicer NOVA_RNAi Explained (Interactive) http://www.pbs.org/wgbh/nova/body/rnai-explained.html Explained: RNA Interference

Yes

No

RISC RNA Interference (Nature Reviews Genetics 5 min video) Video on YouTube http://www.youtube.com/watch?v=cK-OGB1_ELE Video on Nature.com http://www.nature.com/nrg/multimedia/rnai/animation/index.html

Yes

RNAi An Introduction to Reverse Genetic Tools Gene Silencing RNA Interference (RNAi): a new pioneering discovery

No

No

siRNA NOVA_The RNAi Cure Gene-silencing drug can halt and reverse deadly brain disorder

No No







53


Name ______________________________ Period________ Date__________________ Group 1: Argonautes

Directions. Watch the interactive slide show, Learn More About RNAi, and answer the following questions. http://www.nature.com/nrg/multimedia/rnai/animation/index.html 1. What does it mean to “silence” a gene? 2. What enzyme is responsible for the transcription of DNA into primary mRNA? 3. What is the purpose of the ribosomes in the cytoplasm? 4. What must RNAi target to silence genes? 5. What is the difference between exogenous and endogenous siRNA? 6. What is a major use of siRNA in the research laboratory? 7. What is the function of dicer? 8. What is the RISC complex? What does it do? 9. What part of the miRNA must match the mRNA? Why is this important?


54

Silence of the Genes Lesson #2 Who Put the “I” in my RNA? Part 1

Name ______________________________ Period _______ Date___________________ Group #2: C. elegans

Directions. Read the two articles and answer the following questions. Discovery of RNA Interference 1. Who discovered RNAi? 2. How was RNAi discovered? 3. Why is the discovery of RNAi important (give 2 reasons) 4. What are some proposed medical uses of RNAi? 5. What are some of the drawbacks of using RNAi? NOVA_RNAiExpert Q & A 1. Who is the expert in this interview? 2. How do plant viruses protect themselves against RNAi’s? 3. Does RNAi therapy target the patient’s genes or the viral genes? 4. Does RNAi change the genetic sequence? 5. Currently, what is the biggest obstacle to using RNAi technology to treat disease?

55


Name ______________________________ Period _______ Date___________________ Group #3: Dicers

Directions. Watch the RNAi Explained Interactive Slide Show and read the article Explained: RNA Interference. Answer the questions associated with each resource. RNAi Explained (http://www.pbs.org/wgbh/nova/body/rnai-explained.html) 1. How does a cell recognize viral RNA? 2. What is RNAi? What is its purpose in the cell? 3. What is dicer? 4. What is RISC? 5. How does the RISC/RNA complex prevent protein production? Explained: RNA Interference 1. What is siRNA? 2. Compare/contrast siRNA and microRNA.


56


Name ______________________________ Period _______ Date___________________ Group #4: RISC

Directions. Watch the video, RNA Interference, and answer the following questions. You can find the video on YouTube (http://www.youtube.com/watch?v=cK-OGB1_ELE) or Nature Reviews Genetics(http://www.nature.com/nrg/multimedia/rnai/animation/index.html) 1. What is RNAi? 2. What is siRNA? How is it delivered into the cell? 3. What endonuclease cuts siRNA and microRNA into smaller segments? 4. How large are most siRNAs and microRNAs? 5. What happens to the siRNA when it binds to the Argonaute protein? 6. The combination of siRNA, Argonaute protein, and other proteins make up what

structure? 7. What is the function of the RISC complex? 8. What part of the microRNA pairs with the target mRNA? 9. Which type of small RNA-siRNA or microRNA- can pair with more types of mRNA? Why? 10. Argonaute and their small RNA cofactors are found in what type of organisms?



57


Name ______________________________ Period _______ Date___________________ Group #5: RNAi

Directions. Read the two papers and answer the following questions. An Introduction to Reverse Genetic Tools 1. What is difference between forward and reverse genetics? 2. What is the goal of reverse genetics? 3. In what model organism was RNAi first discovered? Gene Silencing RNA Interference (RNAi): a new pioneering discovery 1. What is RNA interference? 2. What is siRNA and what is its function? 3. How many base pairs is siRNA? 4. What is the RISC complex? 1. 5. At the end of the article, the author gives examples of how gene silencing can be used in

plant research and development. Describe the one you find the most interesting.

58


Name ______________________________ Period _______ Date___________________ Group #6: siRNA

Directions. Read the two papers and answer the following questions. The RNAi Cure 1. What are the some of the obstacles to using RNAi technology to treat disease? 2. Describe how RNAi might be used to treat the following diseases: Macular degeneration Hepatitis C Huntington’s disease HIV Respiratory Infections Cancer

59

Gene-silencing drug can halt and reverse deadly brain disorder (Editorial) 1. What is Huntington’s disease? 2. How is RNAi being used to treat Huntington’s disease?

60


Name ______________________________ Period _______ Date___________________ Group ______________________________

Directions. You will now be placed into another group (Group 1, 2, 3, or 4). Each group will contain a member of each of the expert groups (Argonautes, C. elegans, Dicers, RISC, RNAi’s, siRNAs). Discuss what you learned in your Expert group. Some of you will have the same questions; decide as a group how best to answer the question. Some of the questions on this worksheet are brand new. As a group, determine the best way to answer those questions. 1. What is RNAi? 2. Who discovered RNAi? 3. How was RNAi discovered? 4. In what model organism was RNAi first discovered? 5. What does it mean to “silence” a gene? 6. What must RNAi target to silence a gene? 7. Describe 3 uses of RNAi technology. 8. What are FlavrSavr tomatoes? How was gene silencing used to produce these tomatoes? 9. What was the first human disease treated with RNAi? 10. How is RNAi being used to treat Huntington’s disease? 11. How is RNAi being used to treat cancer? 12. What is the difference between forward and reverse genetics?

61

13. What is meant by “off-target” effects of RNA interference? 14. What enzyme is responsible for the transcription of DNA into primary mRNA? 15. What is dicer? What is its purpose? 16. siRNA and miRNA are called “small RNAs”. How small are they (how many nucleotides)? 17. What is the RISC complex? What molecules comprise the RISC complex? What is its

function? 18. What happens to the siRNA when it binds to the Argonaute protein?

19. What part of the siRNA must match the target mRNA? What part of the miRNA must match the target mRNA?

20. Which type of small RNA (siRNA or microRNA) can pair with more types of mRNA? Why? 21. Argonaute and their small RNA cofactors are found in what type of organisms? 22. Does RNAi change the genetic code of a gene? Explain. 23. What are some problems associated with using RNAi to treat disease?

62


Name ______________________________ Period _______ Date___________________ Group #2: C. elegans

Directions. Return to your expert group (Argonautes, C. elegans, Dicers, RISC, RNAi’s, siRNAs ). On your whiteboard, draw a diagram that illustrates the mechanism of RNA interference. At a minimum, be sure to include the following in your diagram: long dsRNA, dicer, siRNA, miRNA, Argonaute, RISC, mRNA. Once you are happy with the diagram on your whiteboard, copy it on this paper.

63

LESSON THREE: “I Said – BE QUIET!!!” KEY QUESTIONS: What makes up a Research Laboratory? Who is Dr. Schultz and what is corneal repair? How do genes get “silenced”? Do you remember the steps to gel-electrophoresis? What is the IACUC? KEY SCIENCE TOPICS: Biotechnology protocols/practices, fundamental cellular and molecular mechanisms, animal husbandry, applied genetics, translational research, animal research protocols. OVERALL TIME ESTIMATES:50 minutes (PowerPoint on Lab Research 10 minutes, Nova video on Gene Silencing 15 minutes, media review 5 minutes, Pipetting by Design activity (lab stations) 20 minutes) LEARNING STYLES: Visual, auditory, kinesthetic VOCABULARY: RNAi: (RNA interference) The process of using small pieces of double-stranded RNA to reduce the activity of specific genes. The process occurs naturally in many organisms and is now commonly used in basic research Macular Degeneration: Loss of centralized vision often due to detachment of the retina from the choroid. Dry form of degeneration forms from accumulation of drusen (cellular debris) detaching retina, wet form caused by accumulating blood vessels which also detach retina. Cornea: a transparent “window” into the eye. Recognized as a-vascular, the cornea is composed of epithelial, stromal, and endothelial tissues. It is the first surface of light refraction in visual acuity. Gel–electrophoresis: Agarose gel electrophoresis is an easy way to separate DNA fragments by their sizes and visualize them. It is a common diagnostic procedure used in molecular biological labs. Adjustable Micropipetting: An adjustable micropipettor is essentially a precision pump fitted with a disposable tip. The volume of air space in the barrel is adjusted by screwing the plunger farther in or out of the piston, and the volume is displayed on a digital readout. Depressing the plunger displaces the specified volume of air from the piston; releasing the plunger creates a vacuum, which draws an equal volume of fluid into the tip. The withdrawn fluid is then expelled by depressing the plunger again. E–Gel cassette: a premade agarose gel slab held within a plastic “cassette” that can be inserted into a compact and movable power source. Agarose: a gelatinous substance derived from the structures of red algae, used as a nutrient media for culturing bacterial plates and as a porous media for gel-electrophoresis (due to its relatively neutral charge). LESSON SUMMARY: The students will be introduced to the research laboratory facilities and personnel of Dr. Gregory Schultz. A PowerPoint will highlight pictures of laboratory equipment/space and a generalization of the corneal tissue repair experimentation currently in progress dealing with gene silencing. In addition, the students will watch a Nova video that takes them on a short journey of the history of gene silencing and its discovery within petunias. Finally, hands-on review of biotechnology techniques will prepare the class for Lesson 4’s DNA extraction and PCR set-up for silk gene amplification. STUDENT LEARNING OBJECTIVES: The student will be able to…

1. List and define gene silencing methodologies. 2. Describe the ingredients of an active research lab. 3. Discuss the merits of empirical evidences in society’s decision making abilities (i.e.drug manufacturing

etc…)

http://en.mimi.hu/biology/rna_interference.html

http://en.mimi.hu/biology/strand.html

http://en.mimi.hu/biology/rna.html

http://en.mimi.hu/biology/organ.html

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4. Give a brief overview of the development of gene silencing and its history. 5. Demonstrate proper pipetting technique and gel- electrophoresis loading. 6. State the proper steps, times, and temperatures for polymerase chain reaction (PCR)

STANDARDS SC.912.L.14.6 SC.912.L.14.50 SC.912.L.15.15 SC.912.L.16.3 SC.912.L.16.6 SC.912.L.16.7 SC.912.L.16.9 SC.912.L.16.10 SC.912.L.16.11 SC.912.L.16.12 SC.912.N.2.4 SC.912.N.2.5 SC.912.N.4.1 SC.912.N.4.2 MATERIALS:

6 stations of biotech review equipment (micro-pipettes, gel chambers/power source, assorted dyes, gels) BACKGROUND INFORMATION: Instructors should be prepared to summarize gene silencing and its history (http://www.pbs.org/wgbh/nova/body/rnai.html). Be familiar with the listed vocabulary. Viewing the PowerPoint and Nova video ahead of time should re-acquaint the instructor to laboratory research and the development of gene silencing for modern biotechnology practices. In addition, a review of the research done by Dr. Schultz will help with a background involving his corneal tissue repair and the use of siRNA’s and miRNA’s. (http://obgyn.ufl.edu/research/schultz/currentresearch.php) ADVANCE PREPARATION:

1. Prepare all copies of review instructions for each student (pipetting by design handout) 2. Prepare PowerPoint for class viewing. 3. Prepare Nova video for class viewing. 4. Setup each of 6 lab stations with a 20µl and a 200µl micropipette, gel chamber/power, gels (pre-made), 4

colors of dyes, and one demo station for PCR set-up, 96 well plates 5. Make copies of discussion questions – Teacher Pages.


1. (10 minutes) Students will view the PowerPoint on Dr. Schultz’ lab/team. 2. (15 minutes) Students will then view the Nova video on Gene Silencing. 3. (5 minutes) Class discussion to summarize both media presentations. Use discussion handout questions. 4. (20 minutes) Lab stations – hands on biotech review (follow paper with instructions).


Students will turn in discussion handout as “ticket out the door” EXTENSIONS: ACTIVITIES: none

http://www.pbs.org/wgbh/nova/body/rnai.html

http://obgyn.ufl.edu/research/schultz/currentresearch.php

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REFERENCES: http://rationalmd.com/cell-biology-glossary/ http://www.cabrini.edu/sepchedna/2002%20Workshop/Labs/measurements_and_micropipetting.htm University of Florida CPET-Pipetting by Design Nova Video: http://www.pbs.org/wgbh/nova/body/rnai.html Thermo Scientific – RNAi video 6 minutes: http://www.nature.com/nrg/multimedia/rnai/animation/index.html http://www.rnaiweb.com/RNAi/RNAi_Web_Resources/ Fire A et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 1998 Feb 19;391(6669):806-11.

http://rationalmd.com/cell-biology-glossary/

http://www.pbs.org/wgbh/nova/body/rnai.html


http://www.rnaiweb.com/RNAi/RNAi_Web_Resources/

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=9486653

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Teacher Pages: Discussion Questions from NOVA Video and Research PowerPoint Key Lead the students through a summarization to clarify video and PowerPoint information. Answer questions during class discussion for future reference: 1. When was the approximate discovery of gene silencing? 1998 by Andrew Fire and Craig Mello

2. What organism has been the primary investigatory specimen? C. elegans

3. What actually causes the “silencing” to occur? a “policing force” (protein Argonaut) recognizes extra

genetic Information and essentially shuts off all genes that are similar including “normal”

4. How can RNAi gain access to the host cell’s genome? (How does it get in?) several methods

allow: ingestion, vector virus’, bacterial plasmids, exogenously induced

5. What benefits could be derived from such technology? many: cancer inhibition, diseases with

accumulating protein compounds, inhibition of growth factors that cause tumor formation

6. What is a primary use of RNAi’s in current genomic research? most often used as a tool for

determing gene purpose (what protein is going to be expressed). Incorporate RNAi – see what “doesn’t” happen

7. In what way was Dr. Schultz’ lab using the silencing techniques? wanted to suppress protein

matrix (collagen etc..) formation in extracellular space within corneal tissue

8. What organization is responsible for much of animal research oversight? IACUC –

Institutional Animal Care Use Committee 9. How is an RNAi different than the “normal” gene/RNA? double stranded, contains both sense

and antisense portions of original gene sequence

10. If given the opportunity, what gene would you really like to turn off? Answers varied

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Student Pages: Discussion Questions from NOVA Video and Research PowerPoint Lead the students through a summarization to clarify video and PowerPoint information. Answer questions during class discussion for future reference: 1. When was the approximate discovery of gene silencing? 2. What organism has been the primary investigatory specimen? 3. What actually causes the “silencing” to occur? 4. How can RNAi gain access to the host cell’s genome? (how does it get in?) 5. What benefits could be derived from such technology? 6. What is a primary use of RNAi’s in current genomic research? 7. In what way was Dr. Schultz’ lab using the silencing techniques? 8. What organization is responsible for much of animal research oversight? 9. How is an RNAi different than the “normal” gene/RNA? 10. If given the opportunity, what gene would you really like to turn off, in any organism?

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Teacher Pages: Pipetting by Design See cpet.ufl.edu for lesson plan.

cpet.ufl.edu

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LESSON FOUR: Let’s See What’s In There KEY QUESTIONS: Why do the metamorphic stages of silkworms differ if they have the same DNA? What is PCR and how is it used in science/biotechnology? What are the components and steps to PCR? Why is amplification of genetic material needed? KEY SCIENCE TOPICS: Biotechnology protocols/practices, fundamental cellular and molecular mechanisms, animal husbandry, applied genetics, translational research, animal research protocols. OVERALL TIME ESTIMATES: 90 minutes (5 minutes lesson overview, 30 minutes DNA extraction, 10 minutes PCR set-up, 5 minutes conclusion) LEARNING STYLES: Visual, auditory, kinesthetic VOCABULARY: Thermocycler: A laboratory apparatus used to amplify segments of DNA via the polymerase chain reaction (PCR) process. Microcentrifuge: An apparatus consisting essentially of a compartment spun about a central axis to separate contained materials of different densities or to separate colloidal particles suspended in a liquid. Amplification: An increase in the number of copies of a gene in a cell, resulting in an elevation in the level of the RNA or protein encoded for by the gene and a corresponding amplification of the phenotype that the gene confers on the cell. Drug resistance in cancer cells is linked to amplification of the gene that prevents absorption of the chemotherapeutic agent by the cell. Denaturation: A process in which the structure of nucleic acid is disrupted, such as the dissociation of a double stranded DNA into a single stranded state by heating. Annealing: To recombine (nucleic acids) at low temperature after separating by heat. Extension: The addition of nucleotides to starter primers on a desired gene for amplification. Transilluminator: The passing of a light through the walls of a body part or organ to facilitate internal inspection. LESSON SUMMARY: Students will follow instructions on DNA extraction for each of 3 samples of silkworm metamorphic stages (egg, larva, pupa, adult). PCR will be used to amplify silk and membrane genes extracted from each of the metamorphic samples. These extractions will then be electrophoresed and used as a control against the genes that will be hypothetically “silenced” (during our Lesson #7 analysis). STUDENT LEARNING OBJECTIVES: The student will be able to…

1. List and Describe DNA extraction techniques. 2. Explain the purpose of primers. 3. List the steps of PCR while explaining the scientific basis for each step. 4. Illustrate a flow chart/pathway from DNA extraction to electrophoresis.

STANDARDS SC.912.L.14.2 SC.912.L.14.3 SC.912.L.16.4 SC.912.L.16.5

http://en.wikipedia.org/wiki/Laboratory

http://en.wikipedia.org/wiki/DNA

http://en.wikipedia.org/wiki/Polymerase_chain_reaction

http://www.biology-online.org/dictionary/Nucleic_acid

http://www.biology-online.org/dictionary/Dissociation

http://www.biology-online.org/dictionary/DNA

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SC.912.L.16.6 SC.912.L.16.9 SC.912.L.16.10 SC.912.L.16.12 SC.912.N.1.7 MATERIALS:

6 Lab Stations of 4 students each.

Lab materials as listed on Teacher page: DNA extraction; and Teacher Page: PCR

1 copy Teacher Page: DNA extraction instruction page

1 copy Teacher Page: PCR amplification instruction page

1 copy per student DNA extraction instruction page (student)

1 copy per student PCR amplification instruction page (student) BACKGROUND INFORMATION: Students will use each of the metamorphic stages of the silkworm life cycle as DNA samples. The purpose of using samples from each cycle is to demonstrate that though phenotypic expression is altered with each metamorphic stage (most dramatically), they still contain the same DNA genome. Students will use their silkworm samples to compare two specific genes: the P25 silk gene and a membrane transport protein. These two specific genes have been sequenced and identified. Primers are used to initiate amplification of these genes during the Polymerase Chain Reaction (PCR). The amplified genes will then be segregated during gel-electrophoresis by passing the DNA fragments through an agarose gel. DNA is negatively charged (due to the phosphate groups), and will thus migrate through the gel toward the positive electrode. Fragment size restricts passage speed so the smaller fragments will travel toward the electrical charge more quickly and separate from the larger fragments. All of the metamorphic samples should demonstrate equal fragment travel (banding), regardless of what stage as they all of consistent DNA. These PCR samples will then be used as the control samples against the experimental group containing the “silenced” silk gene. The membrane protein will not be altered to add to the comparison and give further representation that fragment distances were a result of RNAi influences and not other unknown factors. ADVANCE PREPARATION:

1. Prepare copies of Teacher Pages for DNA extraction and PCR, DNA gel-electrophoresis, and student lab instruction handouts

2. Prepare list of solutions Teacher Page: “Lab Set-up” 3. Setup each of 6 lab stations with list of materials from Teacher Page: “Lab Set-up”


1. (5 minutes) Lesson Overview – prelab instructions. Instructor will go over the goal of the lesson as well as quickly verbalizing the primary points of the lab (from the lab handout), checking for understanding from the students.

2. (30 minutes) DNA extractions – students will perform 3 of 4 metamorphic stages (egg, larva, pupa, adult). Each lab group will select 3 of the 4 metamorphic stages for extraction, this is to simply produce variety of extraction throughout the class audience, and reduce amount of material / time. With 6 lab groups all stages will be covered (can be written on classroom white board to make sure all stages are being extracted).

3. (10minutes) PCR set-up, run (2 hours total time, students out of class). After following instructions at lab station, students will place their samples within the thermocycler for amplification. Because this runs for 40 cycles, the students will collect their amplifications at the next lesson.

4. (5 minutes) Lesson summary – ‘ticket out the door” Brainstorming activity to list on board: both practical and fantastical applications (to be used later in research lesson). Students will share ideas of application potential, many of these will be written on the white board. In addition, the students will have an index card to write down 3 “ideas” (some may come from white board, especially from those students who are creatively challenged).

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Check each of six stations for progress and correct implementation

Students will write down 3 applications from brainstorming to show at door (to encourage thought on next project)

EXTENSIONS: ACTIVITIES:

Protein gel-electrophoresis from different silkworm metamorphic cycles, to compare protein expression. See appendix 1 for protocol. This extension would properly illustrate the fact that DNA is conserved in metamorphic stages and proteins are uniquely expressed because of what metamorphic stage the specimen is developing within.

REFERENCES: Vocabulary: www.biology-online.org, www.freedictionary.comhttp://en.wikipedia.org, http://www.thefreedictionary.com/transillumination http://en.wikipedia.org/wiki/Thermocycler Sigma DNA Extraction and PCR Kit: http://www.sigmaaldrich.com/catalog/product/sigma/xnat2?lang=en&region=US,




http://www.thefreedictionary.com/transillumination

http://en.wikipedia.org/wiki/Thermocycler

http://www.sigmaaldrich.com/catalog/product/sigma/xnat2?lang=en&region=US

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Student Pages: Silkworm DNA Extraction / PCR DNA Extraction

1. Label a screw top micro centrifuge tube with the sample type and an identifying mark for your group. Repeat for your remaining samples. You will repeat each of the following steps for each sample that you run.

2. Pipette 100 mol of Extraction Solution (ES) into a screw top micro centrifuge tube. 3. Add 25 mL of Tissue Preparation Solution (TPS) to the tube and pipette up and down to

mix. 4. Rinse the scalpel and forceps in ethanol prior to use and between different samples.

Place a 2–10 mg piece of tissue into the solution and close the tube. Mix thoroughly by flicking the tube. Ensure the tissue is in the solution.

5. Incubate sample at room temperature for 10 minutes. 6. Incubate sample at 95°C in the heat block for 3 minutes. Be careful, the heat block is

VERY hot! 7. Add 100 mL of Neutralization Solution B (NSB) to sample and mix by flicking the tube. 8. Use DNA sample immediately for PCR.

PCR

1. Label a PCR tube with the sample type and an identifying mark for your group. Repeat for your remaining samples.

2. Half of the groups will use primer sets 1, and the other half will use primer sets 2. Wait for your instructor to indicate which set your group will run, and label your tubes with a 1 or 2, accordingly.

3. Add the following reagents to your PCR tube: Reagent Volume

PCR Master Mix (reaction mix, primers, water)

16µl

DNA sample 4 µl

4. Take your PCR tubes over to the thermocycler to run through the temperature cycles.

Once the cycles have run, the amplified DNA will be kept cold, at 4˚C until ready for gel electrophoresis.

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Student Pages: Silkworm DNA Gel Electrophoresis DNA Gel Electrophoresis

1. Plug red E-Gel PowerBase™ into an electrical outlet. 2. Remove gel cassette from package. 3. Insert the gel (with comb in place) into the base right edge first. The Invitrogen logo

should be located at the bottom of the base. Press firmly at the top and bottom to seat the gel cassette in the E-Gel PowerBase™. A steady, red light will illuminate if the gel cassette is correctly inserted.

4. Remove and discard comb from the E-Gel® cassette. 5. Add 10μl sterile distilled H2O to wells 4-8. 6. Add 20μl sterile distilled H2O to wells 1-3, 9-12. 7. Add 8μl DNA samples to wells 5-8. 8. Add 8μl marker to well 4.

9. Press and release the 30 minute button on the E-Gel® PowerBase™ to begin

electrophoresis. 10. At the end of the run, the current will automatically shut off and the power base will

display a flashing red light and beep rapidly. Press either button to stop the beeping, and unplug the E-Gel® PowerBase™.

11. Remove the gel cassette and analyze your results by viewing on one of the transilluminators.

Well #

1 2 3 4 5 6 7 8 9 10 11 12

What to

add to

the well

20μl water

20μl water

20μl water

10μl water

+ 8μlmarker

10μl water

+ 8μl

DNA

10μl water

+ 8μl

DNA

10μl water

+ 8μl

DNA

20μl

water

20μl water

20μl water

20μl water

20μl water

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Student Pages: Silkworm Protein Extraction and Electrophoresis Extraction

1. Label one 1.5 ml flip top micro tube for each of the silkworm life stage samples. Also label one screw cap microtube for each sample.

2. Add 250μl of Laemmli sample buffer (blue liquid, labeled LB) to each labeled flip top microtube.

3. Cut a piece of each silkworm life stage sample about 0.25 x 0.25x 0.25 cm3 and transfer each piece into a labeled flip top micro test tube. Close the lids.

4. Flick the microtubes 15 times to agitate the tissue in the sample buffer. 5. Incubate for 5 minutes at room temperature. 6. Carefully transfer the buffer by pouring from each flip top microtube into a labeled

screw cap microtube. Do not transfer the silkworm! 7. Heat the samples in screw cap microtubes for 5 minutes at 95°C in the heat block. Be

careful – heat block is very hot!

Gel Electrophoresis 1. Your instructor has set up the gel boxes with 15% polyacrylamide gels, with a 1% TGS

buffer as the electrophoresis running buffer. 2. Load your gel using the p20 pipette and the special Protein Gel Pipette tips (available

from your instructor) as follows: Lane Volume Sample

1 Empty Empty

2 Empty Empty

3 Empty Empty

4 5 µl Protein Standards

5 10 µl Silkworm sample 1



8 10 µl Silkworm sample 4 (or empty)

9 Empty Empty

10 Empty Empty

3. Electrophorese for 30 minutes at 200 V in 1x TGS electrophoresis buffer. 4. Remove gel from gel cassette by removing the cover sticker and slowly and gently

sliding the gel out in a gel tray with purified water. Gently swish your gel in the water. 5. Pour out water and rinse once more with water. Pour out water. 6. Add 25 ml of Coomassie Blue safe stain to your gel tray. Let sit for at least 1 hour. 7. Pour out gel stain and rinse with water. Let sit for at least 1 hour. 8. View gels.

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Teacher Pages: PowerPoint on Silkworm PCR / Electrophoresis Silkworm PCR & Gel Electrophoresis

Introduction to PCR

1983 Kary Mullis (Cetus Corp) developed the molecular biology technique that has revolutionized genetic research:

The Polymerase Chain Reaction

PCR quickly transformed molecular biology into a multidisciplinary research field

Introduction to PCR

Object of PCR: produce a large amount of DNA in a test tube (in vitro) starting with only a trace amount

Controlled enzymatic amplification of a DNA sequence, or gene, of interest

Tiny amounts of genomic DNA from a drop of blood, single hair, or cheek cell can generate enough DNA to study

Introduction to PCR

PCR impacted several areas of genetic research: o used as a medical diagnostic tool to detect specific mutations that may cause genetic

disease o used in criminal investigations and courts of law to identify suspects o used in the sequencing of the human genome

Introduction to PCR

Prior to PCR, the use of molecular biology techniques for therapeutic, forensic, pharmaceutical, agricultural, or medical diagnostic purposes was neither practical nor cost-effective

The development of PCR transformed molecular biology from a difficult science to one of the most accessible and widely used disciplines of biotechnology

PCR amplification

With PCR, you can target and make millions of copies (amplify) a specific piece of DNA (or gene) out of a complete genome

In our experiment, you will amplify a region within your own chromosome 16

PCR amplification

PCR makes use of the same basic processes that cells use to duplicate their DNA (replication) o Complementary DNA strand hybridization o DNA strand synthesis via DNA polymerase

PCR amplification

Recipe for a PCR amplification of DNA: o DNA template: Your DNA, containing the intact sequence of DNA to be amplified o Deoxynucleotides: raw material of DNA o DNA polymerase: enzyme that assembles the nucleotides into a new DNA chain

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o Magnesium ion (Mg2+): cofactor (catalyst) required by DNA polymerase to create the DNA chain

o Primers: pieces of DNA complementary to the template that tell DNA polymerase exactly where to start

o Salt buffer: provides the optimum ionic environment and pH for the PCR reaction

PCR amplification

PCR: three main steps o Denaturation (94 degrees, 1min) o Annealing (60 degrees, 1min) o Extension (72 degrees, 2min)

40 cycles Amplified exponentially Results in 1.1x1012 sets of precise-length DNA

o Gel loading tips

Always use a clean pipette tip prior to drawing sample

Push the plunger to the first stop to push air or sample out of the pipette

Place your tip into the well and expel the sample without jabbing the tip through the bottom of the gel (this will cause a hole to form at the bottom of the well, allowing your DNA to leak out)

Gel electrophoresis

Migration of charged particles under the influence of an electric field o DNA is charged o Moves toward electrode

Gel electrophoresis

Horizontal gel made of agarose o Agarose is a substance extracted from seaweed, similar to gelatin o Different size fragments of DNA move through the agarose gel at different speeds due

to the sieving action of agarose

DNA exposed

Fragments of DNA are separated on the gel revealing bands or lines

Use dye and UV light to see the bands

See bands of different sizes which represent fragments of DNA

This will not tell us the DNA sequence

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LESSON FIVE: Who to Choose? – Speak Up!!

KEY QUESTIONS: What is a UTR? Which codon “starts” the reading sequence in mRNA? What is meant by RefSeq in siRNA selections? What is the purpose of BLAST? KEY SCIENCE TOPICS: Biotechnology protocols/practices, fundamental cellular and molecular mechanisms, animal husbandry, applied genetics, translational research, animal research protocols. OVERALL TIME ESTIMATES: 50 minutes (siRNA/RNAi selection web site instruction 25 minutes, Gel-electrophoresis of amplified silk and membrane transport genes 30 minutes (total), prep mulberry leaves with ingestible RNAi’s remaining time) LEARNING STYLES: Visual, auditory, kinesthetic VOCABULARY: 5’ UTR: The five prime UnTranslatedRegion (5' UTR), can contain elements for controlling gene expression by way of regulatory elements. It begins at the transcription start site and ends one nucleotide (nt) before the start codon (usually AUG) of the coding region. In prokaryotes, the 5' UTR usually contains a ribosome binding site (RBS), also known as the Shine Dalgarno sequence (AGGAGGU). NCBI: National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov) – advances science and health by providing genomic information. BLAST: The Basic Local Alignment Search Tool (BLAST- http://www.ncbi.nlm.nih.gov/BLAST/Blast.cgi ) finds regions of local similarity between sequences. The program compares nucleotide or protein sequences to sequence databases and calculates the statistical significance of matches. BLAST can be used to infer functional and evolutionary relationships between sequences as well as help identify members of gene families. Codon: a triplet sequence of nucleotides that directs the tRNA’s ”anticodon” to acquire select amino acids for protein production. Poly-A tail: Polyadenylationis the addition of a poly(A) tail to an RNA molecule. The poly(A) tail consists of multiple adenosine monophosphates; in other words, it is a stretch of RNA that has only adenine bases. In eukaryotes, polyadenylation is part of the process that produces mature messenger RNA (mRNA) for translation LESSON SUMMARY: Students will use web-based information to learn the methodologies associated with RNAi selection. A step-wise process is also found on a student handout that will guide them in the process of selection (even without the web sites). However, all RNAi selections must be performed using the BLAST protocols and sequence comparisons. In addition, the students will use the amplified genes from the previous lesson’s DNA extractions and run gel-electrophoresis to segregate gene “bands”. Gels will be run at the beginning of the lesson to allow web use during “down time” of the electrophoresis units. Finally, students will apply the ingestible RNAi’s to the silkworm leaves to gain transfection of RNAi into the host’s cellular spaces. STUDENT LEARNING OBJECTIVES: The student will be able to…

1. Analyze and interpret the process of RNAi selection, using web-based tools 2. Perform gel-electrophoresis on amplified select genes. 3. Explain the uses of BLAST. 4. Suggest reasoning for RNAi selection failures (undesirable results).

http://en.wikipedia.org/wiki/Gene

http://en.wikipedia.org/wiki/Regulatory_element

http://en.wikipedia.org/wiki/Transcription_start_site

http://en.wikipedia.org/wiki/Nucleotide

http://en.wikipedia.org/wiki/Start_codon

http://en.wikipedia.org/wiki/Coding_region

http://en.wikipedia.org/wiki/Prokaryotes

http://en.wikipedia.org/wiki/Ribosome_binding_site

http://en.wikipedia.org/wiki/Shine_Dalgarno


http://www.ncbi.nlm.nih.gov/BLAST/Blast.cgi

http://en.wikipedia.org/wiki/RNA

http://en.wikipedia.org/wiki/Adenosine_monophosphate

http://en.wikipedia.org/wiki/Adenine

http://en.wikipedia.org/wiki/Eukaryote

http://en.wikipedia.org/wiki/Messenger_RNA

http://en.wikipedia.org/wiki/Translation_(biology)

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STANDARDS SC.912.L.14.2 SC.912.L.14.3 SC.912.L.15.15 SC.912.L.16.4 SC.912.L.16.5 SC.912.L.16.6 SC.912.L.16.9 SC.912.L.16.10 SC.912.L.16.12 SC.912.N.1.3 SC.912.N.1.4 SC.912.N.1.5 SC.912.N.1.6 SC.912.N.1.7 MATERIALS:

1 copy of Teacher Pages: How To Choose Silencing Targets (2 pages)

1 copy of Teacher Pages: Instructions on Silkworm DNA gel-electrophoresis

Computer cart: 1 computer / student

Select RNAi and mulberry leaves BACKGROUND INFORMATION: Due to the complexity of this lesson, the instructor is encouraged to run through RNAi selection processes several times to gain a greater confidence before presenting to students. This lesson is an introduction to the methodologies associated with selection of certain silencing sequences (but is not exhaustive). With these things in mind, it is quite possible that students will not walk away fully versed in the process (most certainly not after only a few attempts.) Yet, a basic understanding can be acquired and hold value to the overall unit. The silkworm gene for silk is known, and thus, an RNAi was built and acquired (as a means to silence the silk gene). Students can be shown how this process was completed. The final portion of the lesson allows the students to work with the silkworms. The RNAi will be applied to the food source in the hopes that the larva will ingest the silencing mechanism. It is unclear when and if transfection will occur, therefore, application will be induced for each instar stage of larval development to gain adequate infiltration. ADVANCE PREPARATION:

1. Prepare 6 lab stations (4 students each) with list of materials found on Teacher Pages: Instructions on Silkworm DNA gel-electrophoresis

2. Setup computer stations from computer cart allowing for wireless Internet access. Write website on board for RNAi selection location http://www.rnaiweb.com/RNAi/RNAi_Glossary/index.html

3. Setup location for RNAi and mulberry leaves 4. Make copies of Teacher Pages: How to Choose Silencing Techniques and Gel-Electrophoresis (1/student) 5. List several genes on board for student use during web access. (Perform selection process in advance)


1. (10 minutes) Students will prep DNA gel-electrophoresis with amplified silk gene/membrane transport gene and begin running gels. Wells will include a DNA standard 1kb marker and each of 3 metamorphic stages. Gels will run for 30 minutes.

2. (25 minutes) Students will have Internet access to the RNAi selection website. Following the Silencing Techniques handout, students will perform several examples of instructor pre-selected genes (listed on board). Encourage students to “hang in there” as they may become frustrated, provide “cues” as they work through the processes. Remind students to use handout to stay on target. This portion will be done as a webquest, it may take additional class time or can perhaps be used as an extension out of class)

http://www.rnaiweb.com/RNAi/RNAi_Glossary/index.html

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3. (5 minutes) When gel-chamber sounds off (beeps after 30 minutes), power supply turns off and gels are complete. Students will remove cassettes and view their bands under the UV transilluminator. Comparisons of bands can then be made and analysis of gene consistency (against each metamorphic cycle) can be noted on directions page.

4. (10 minutes) Mulberry leaves will be prepped with RNAi for silkworm larva ingestion. ASSESSMENT SUGGESTIONS:

Students will present an “exit card” (see Appendix #2) that poses a gel electrophoresis question that must be answered from their own investigation. Students will turn in student handout with accompanying diagram and questions. Check for completion and understanding of procedure results.

Oversee RNAi application to mulberry leaves for thoroughness. EXTENSIONS:

Students can produce a standard curve based on the DNA molecular weight marker from well #4 REFERENCES: Vocabulary: www.biology-online.org, www.freedictionary.comhttp://en.wikipedia.org Cenik C, Derti A, Mellor JC, Berriz GF, Roth FP. (2010). "Genome-wide functional analysis of human 5′untranslated region introns.". Genome Biology11 (3): R29. PMID20222956. http://genomebiology.com/2010/11/3/R29/abstract.

Lodish, et al.. "chapter 4.2". Molecular Cell Biology (5th ed.). p. 113. Cenik, C, et al. (2011). "Genome analysis reveals interplay between 5' UTR introns and nuclear mRNA export for

secretory and mitochondrial genes.". PLoS Genetics7 (4). DOI:10.1371/journal.pgen.1001366.

http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1001366




http://en.wikipedia.org/wiki/PubMed_Identifier

http://genomebiology.com/2010/11/3/R29/abstract

http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1001366

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I, the copyright holder of this work, release this work into the public domain. This applies worldwide. In some countries this may not be legally possible; if so: I grant anyone the right to use this work for any purpose, without any conditions, unless such conditions are required by law.

http://www.ncbi.nlm.nih.gov/BLAST/Blast.cgi?CMD=Web&LAYOUT=TwoWindows&AUTO_FORMAT=Semiauto&ALIGNMENTS=50&ALIGNMENT_VIEW=Pairwise&CLIENT=web&DATABASE=nr&DESCRIPTIONS=100&ENTREZ_QUERY=%28none%29&EXPECT=10&FILTER=L&FORMAT_OBJECT=Alignment&FORMAT_T

http://en.wikipedia.org/wiki/en:public_domain

http://upload.wikimedia.org/wikipedia/commons/b/ba/MRNA_structure.svg

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Student Page: Use as a WebQuest (See Appendix #1) siRNA Design Rules for selecting siRNA targets on mRNA sequences:

1. Targets should be located 50-100 nt downstream of the start codon (ATG). 2. Search for sequence motif AA(N19)TT or NA(N21), or NAR(N17)YNN, where N is any

nucleotide, R is purine (A, G) and Y is pyrimidine (C, U). 3. Target sequences should have a G+C content between 35-60%. 4. Avoid stretches of 4 or more nucleotide repeats. 5. Avoid 5'UTR and 3'UTR, although siRNAs targeting UTRs have been shown to

successfully induce gene silencing. 6. Avoid sequences that share a certain degree of homology with other related or

unrelated genes. How to obtain a mRNA or cDNA sequence for target selection: Before picking siRNA target on the gene you choose from the list on the board, first you have to obtain its mRNA sequence from a nucleic acids database or sequence accession number as some siRNA design tools can take accession number as input. It is recommended to use the gene's RefSeq from NCBI, since the RefSeq represents non-redundant, curated and validated, thus most correct, sequences. RefSeq mRNA sequences have unique accession numbers which start with NM or XM, followed by 6 digits. For example, NM_123456 (curated mRNA sequence) or XM_0123456 (model mRNA sequence predicted by genome sequence analysis). There are several ways of searching and retrieving a RefSeq.

1. Search the NCBI Gene database (Entrez Gene) by gene name or symbol at http://www.ncbi.nlm.nih.gov/gene/ and select the right gene of desired organism, go to the page of the gene, scroll down to the "mRNA and Protein(s) " section and look for mRNA sequences with a accession number started with NM or XM.

2. Search Nucleotide database using Entrez query tool at http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Nucleotide and use Entrez Limits settings to restrict your query to the RefSeq database only

o select "RefSeq" from the "Only from" menu, this restricts the query to the RefSeq collection

o select "mRNA" from the "Molecule" menu, this restricts the query to mRNA RefSeq records

Homology search The siRNA targets on the mRNA sequence of a gene should not share significant homology with other genes or sequences in the genome, therefore, homology search is essential for preventing off-target effects. Although most siRNA design tools provide BLAST search option, some simply use NCBI's BLAST tools which sometimes are quite slow. Here are some BLAST tools for homology search.

http://www.ncbi.nlm.nih.gov/RefSeq/


http://www.ncbi.nlm.nih.gov/LocusLink/

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Nucleotide

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NCBI Blast tool: Nucleotide-nucleotide BLAST (blastn) or Search for short, nearly exact matches

Blat tool on UCSC Genome Website http://genome.ucsc.edu/cgi-bin/hgBlat Ensembl Blast http://www.ensembl.org/Multi/blastview

Examples of RNAi target selection

References

1. Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature. 2001 May 24;411(6836):494-8.

2. Elbashir SM, Lendeckel W, Tuschl T. RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev. 2001 Jan 15;15(2):188-200.

3. Reynolds A, Leake D, Boese Q, Scaringe S, Marshall WS, Khvorova A. Rational siRNA design for RNA interference. Nat Biotechnol. 2004 Mar;22(3):326-30.

4. Tuschl Lab, The siRNA user guide.

http://www.ncbi.nlm.nih.gov/BLAST/Blast.cgi?CMD=Web&LAYOUT=TwoWindows&AUTO_FORMAT=Semiauto&ALIGNMENTS=50&ALIGNMENT_VIEW=Pairwise&CLIENT=web&DATABASE=nr&DESCRIPTIONS=100&ENTREZ_QUERY=%28none%29&EXPECT=10&FILTER=L&FORMAT_OBJECT=Alignment&FORMAT_T

http://www.ncbi.nlm.nih.gov/BLAST/Blast.cgi?CMD=Web&LAYOUT=TwoWindows&AUTO_FORMAT=Semiauto&ALIGNMENTS=50&ALIGNMENT_VIEW=Pairwise&CLIENT=web&DATABASE=nr&DESCRIPTIONS=100&ENTREZ_QUERY=%28none%29&EXPECT=1000&FORMAT_OBJECT=Alignment&FORMAT_TYPE=HTM

http://www.ncbi.nlm.nih.gov/BLAST/Blast.cgi?CMD=Web&LAYOUT=TwoWindows&AUTO_FORMAT=Semiauto&ALIGNMENTS=50&ALIGNMENT_VIEW=Pairwise&CLIENT=web&DATABASE=nr&DESCRIPTIONS=100&ENTREZ_QUERY=%28none%29&EXPECT=1000&FORMAT_OBJECT=Alignment&FORMAT_TYPE=HTM

http://genome.ucsc.edu/cgi-bin/hgBlat

http://www.ensembl.org/Multi/blastview

http://www.rockefeller.edu/labheads/tuschl/sirna.html

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Student Pages: Silkworm DNA Gel Electrophoresis Worksheet

List of Materials: red E-Gel PowerBase™ gel cassette 20μl micropipette Micropipette tips 50 ml Distilled water Transilluminator Analysis: Draw a representative line in each lane to show approximate band position in original gel slab. Was there band consistency for each metamorphic cycle? What might account for any differences in bands?

DNA Gel Electrophoresis

1. Plug red E-Gel PowerBase™ into an electrical outlet. 2. Remove gel cassette from package. 3. Insert the gel (with comb in place) into the base right edgefirst. The Invitrogen logo should be

located at the bottom of the base. Press firmly at the top and bottom to seat the gel cassette in the E-Gel PowerBase™. A steady, red light will illuminate if the gel cassette is correctly inserted.

4. Remove and discard comb from the E-Gel® cassette. 5. Add 10μl sterile distilled H2O to wells 4-8. 6. Add 20μl sterile distilled H2O to wells 1-3, 9-12. 7. Add 8μl DNA samples to wells 5-8. 8. Add 8μl marker to well 4.

9. Press and release the 30 minute button on the E-Gel® PowerBase™ to begin

electrophoresis.

Well #

1 2 3 4 5 6 7 8 9 10 11 12

What to

add to

the well

20μl water

20μl water

20μl water

10μl water

+ marker

10μl water

+ 8μl

DNA

10μl water

+ 8μl

DNA

10μl water

+ 8μl

DNA

10μl

water +

8μl DNA

20μl water

20μl water

20μl water

20μl water

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10. At the end of the run, the current will automatically shut off and the power base will display a flashing red light and beep rapidly. Press either button to stop the beeping, and unplug the E-Gel® PowerBase™.

11. Remove the gel cassette and analyze your results by viewing on one of the transilluminators.

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LESSON SIX: The Proposal KEY QUESTIONS: What are some possible applications of RNAi technology? What is the best way to introduce siRNA or miRNA into an organism? In what organisms is RNA interference the most feasible? How does one design a “Glog”?

OVERALL TIME ESTIMATES: Lesson six should take four or five 50 minute class periods. Class #1: Internet research. Class #2: Finish up research; type up formal proposal. Class #3: Create Glog on glogster.com*. Class #4: Student Proposal Presentations *If students have never used Glogster.com, it will probably take two class periods to develop their glogs. LEARNING STYLES: Visual and auditory VOCABULARY: none LESSON SUMMARY: The purpose of this lesson is to introduce students to the process of writing a proposal for scientific research. They will then present their proposals to the class (scientific community) using Glog they have created on Glogster.com (http://edu.glogster.com/). The artifact of the lesson is that the students are University of Florida graduate students interested in studying RNA interference. Students will need to use the knowledge they have gained in the previous lessons to develop an outline of an experiment using RNAi. Students will need to first identify the problem, how they are going to study the problem, what model organism to use, and possible applications of their results. STUDENT LEARNING OBJECTIVES: The student will be able to…

1. Identify a current scientific problem and suggest solutions using RNA interference. 2. Organize, develop, and present a research proposal. 3. Use technology to effectively present a report.

STANDARDS: SC.912.L.14.6 SC.912.L.16.3 SC.912.L.16.5 SC.912.L.16.10 SC.912.L.18.4 SC.912.N.1.1 SC.912.N.1.2 SC.912.N.1.4 SC.912.N.1.6 SC.912.N.1.7 MATERIALS:

Computers with internet access and printer

BACKGROUND INFORMATION: Most students do not have a good understanding of the preparation needed to conduct the experiments they read and hear about from their instructors. Also, high school students usually have not had much experience designing a complex research project. The purpose of this lesson is to give them a taste of the work involved in conducting scientific research. While the format of research proposals can vary somewhat, there are some elements that are common to them all. Proposals generally consist of (1) a concise, descriptive title, (2) an abstract or summary, (3) a problem statement, (4) some initial literature review to show the need for


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the research, (5) a description of the methods used to tackle the problem-the actual experiment, (6) what type of data will be collected, and (7) a conclusion or discussion that summarizes the proposed study. ADVANCE PREPARATION:

1. Make copies of student worksheets. 2. Arrange for computers with student access.

PROCEDURE AND DISCUSSION QUESTIONS WITH TIME ESTIMATES: none ASSESSMENT SUGGESTIONS:

Grade research proposals and presentations. EXTENSIONS: ACTIVITIES: none REFERENCES: Erren, Thomas C., and Philip E. Bourne."Ten Simple Rules for a Good Poster Presentation." National Center for

Biotechnology Information. U.S. National Library of Medicine, 25 May 2007. Web. 12 July 2012. <http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1876493/>.

Faith Cranor, Lorrie."Research Posters 101." XRDS: Article. XRDS, Nov. 1996. Web. 12 July 2012.

<http://xrds.acm.org/article.cfm?aid=332138>. Gendron, Robert P. "Writing the Research Proposal." Writing the Research Proposal. Biology Department, Indiana

University of Pennsylvania, 1 Oct. 2003. Web. 10 July 2012. <http://nsm1.nsm.iup.edu/rgendron/proposal.shtml>.

"Proposal Writing Components." Proposal Writing Components.University of Pittsburgh, Office of Research, n.d.

Web. 12 July 2012. <http://www.pitt.edu/~offres/proposal/propwriting/components.html>. Rodgers, Richard. "Basic Components of a Proposal." UMassAmherst.N.p., 22 June 2003. Web. 10 July 2012.

<http://www.umass.edu/development/foundations/proposal.pdf>.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1876493/

http://xrds.acm.org/article.cfm?aid=332138

http://nsm1.nsm.iup.edu/rgendron/proposal.shtml

http://www.pitt.edu/~offres/proposal/propwriting/components.html

http://www.umass.edu/development/foundations/proposal.pdf

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Silence of the Genes Lesson #6: The Proposal

Name ______________________________ Period _______ Date___________________

Scenario: You are a graduate student at the University of Florida interested in RNA interference research. You have a great idea for your next experiment, but you must get it approved by your scientific advisor and committee. To get approval, you must present a formal proposal that describes what you will do, why you want to do it, how you will do it, and the expected results. Your proposal will consist of two parts: a written proposal (that you will turn into your instructor) and a poster presentation given to your committee. Your poster will be in the form of a “Glog” (http://edu.glogster.com/). You may work either individually or in pairs on this project. What do you need to include your written proposal? 1. Title

Indicate the focus of your research. The title on your proposal does not need to be exactly the same as the title on your poster/Glog.

2. Introduction (1 paragraph) Give a brief description of your research topic or theme.

3. Key Terms/Concepts Define key concepts and terms. Put the definitions in your own words! I don’t want to Google a definition and be taken to Wikipedia!

4. Motivation (1 paragraph) This is the section where you discuss why you want to conduct the research. What is the issue you are addressing? Why is it important? How will it add to current knowledge?

5. Background Research (2-3 paragraphs) You must have at least 3 different resources (remember, that Google is not a resource!). You will need to cite all resources, so make sure you write down where you find the resource. Copy the URL address so you can find the article later.

6. Problem Statement/Description (1 paragraph) This is the “nitty-gritty” of your proposal. This section needs to include a clear and concise description of the purpose of your research. Include not only the results of your specific study, but also the broader applications of your research.

7. Project Description/Experimental Design(1 page) Describe how you are going to conduct the experiment. Will you use siRNA or miRNA? Defend your choice. How are you going to deliver the RNAi drug to the correct target cells? What are your variables? How will you measure your results?

8. Conclusion (1 paragraph) Summarize the main points of your research and what you hope to accomplish.

9. Resources You need at least three resources.


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Link to Glog online

What items need to be on your Glog? 1. Title

Make it short and catchy (but not too cutesy). Save the long title for your written proposal. Be sure to put your title in a central location in an easy to read font.

2. Your Names Write both your first and last names. These should be near your title, but in a smaller font.

3. Background Research Include a brief paragraph (around 5 sentences) that summarizes the research already published on your topic. Don’t make to wordy! You can fill in the details during the poster presentation.

4. Experimental Design Give a brief description of your experiment. You may just list the steps or write a brief description.

5. Picture/photograph of your model organism Let us know what it looks like. Be sure the picture is large enough that it is not fuzzy on your Glog.

6. Key Terms List the major vocabulary used in your proposal. Do not write the definitions on your Glog-you can define them in your presentation.

7. Video clip/Animation Find a video clip/animation to include in your Glog. It needs to be short! (Less than 5 minutes.) Find something interesting that relates to your topic.

http://ademins.edu.glogster.com/research-proposal-glog/

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LESSON SEVEN: Were you Quiet or Not? That, is the Question

KEY QUESTIONS: Can P25 be silenced by ingestible RNAi’s? Do silkworm cocoons exhibit visual/quantitative degrees of silk reduction as a result of gene silencing? Are there other identifiable phenotypic characteristics that were altered by the induction of RNAi to the silkworm diet? Did your initial hypothesis (expected outcome) match the results? KEY SCIENCE TOPICS: Biotechnology protocols/practices, fundamental cellular and molecular mechanisms, animal husbandry, applied genetics, translational research, animal research protocols. OVERALL TIME ESTIMATES: 50 minutes (analysis of outcomes – silk production for cocoon formation) This lesson will ensue following cocooning of silkworm larva – it may take place several days after Lesson #6. The analysis will be performed during the final class period of the unit. LEARNING STYLES: Visual, auditory, kinesthetic VOCABULARY: none LESSON SUMMARY: Students will finalize the silkworm unit with collection of data that includes quantitative measurements and visual assessments of cocoon mass/size based on a mean of similarly altered experimental group cocoons vs. control group cocoon means. A “final lab write-up” will be required and used for assessment – criteria for write-up will be provided to each student with due dates. STUDENT LEARNING OBJECTIVES: At the conclusion of the Silkworm Unit: “Silence of the Genes” The student will be able to…

1. Successfully perform biotechnology practices including: pipetting techniques, DNA extraction, PCR amplification, and Gel-electrophoresis (DNA and protein)

2. Relate the steps of transcription/translation in discussion with fellow classmates. 3. Produce models of proteins from an mRNA nucleotide sequence. 4. Describe the history of gene silencing and its approximate timeline. 5. Deduce and apply procedures for gene silencing selection. 6. Compose a data table to include: the metamorphic life-cycle of Bombyx mori: cycle sequence, approx.

time/cycle, and husbandry requirements, and reproductive requirements/capacities. 7. Predict several practical applications of gene silencing in modern medicine.

STANDARDS SC.912.L.14.2 SC.912.L.14.3 SC.912.L.14.6 SC.912.L.15.15 SC.912.L.16.4 SC.912.L.16.5 SC.912.L.16.6 SC.912.L.16.7 SC.912.L.16.9 SC.912.L.16.10 SC.912.L.16.12 SC.912.L.18.4 SC.912.N.1.1 SC.912.N.1.2 SC.912.N.1.3 SC.912.N.1.4

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SC.912.N.1.5 SC.912.N.1.6 SC.912.N.1.7 MATERIALS:

Primary tools for measurement of larva and cocoons will consist of a digital gram scale, metric rulers, digital photos and visual assessment. Provide one of each for each lab group.

1 copy of Teacher Pages: “Formal Lab Write-up” (make copies to include one/student)

1 copy of Teacher pages: “Data Collection” (make copies to include one/student) BACKGROUND INFORMATION: This final lesson should help the student “tie” all of the investigative lessons together. The analysis verifies the success of the silencing techniques, the learning objectives meet each of the individual lesson primary goals, the completion of the formal write-up will reinforce all components of the investigation and produce much thought / contemplation and perhaps questions. An extension that includes a PCR amplification of the experimental larva would indicate whether or not the silencing influences DNA or mRNA. Band widths may be altered if DNA is influenced, yet no band width changes would imply that mRNA was the effected nucleic acid. This would be a powerful verification for the students to view. As well, no changes in the expression of silk protein (cocoons do not differ) and no band size changes would indicate that the RNAi was not incorporated into the cells (although this is somewhat anticipated – as it is not a proven or known trial). ADVANCE PREPARATION:

1. Make copies of Teacher Pages: Formal Lab Write-up (one copy per student) 2. Make copies of Teacher Pages: Data Collection (one copy per student) 3. Provide measuring tools at each of 6 lab stations: digital scale, digital camera, metric ruler


1. Students will take measurements and visual assessments of all group cocoons. Using the Data Collection handout – students will record results (to be used in formal write-up) and draw illustrations of cocoons to scale. Ask: Are the cocoons visibly smaller, thinner, or does the silk exhibit differences of any kind from the control? Indicate such subject statements such as: color, texture, reflectivity, density of silk, perhaps even unwind to get average lengths of thread.

2. Comparisons of control and experimental cocoon variations will also be assessed and recorded on Data Collection handout. This analysis of data will conclude the study – the entire class period is to be used.


Post Test Assessment (sometime between Lesson #6 and Lesson #7

Formal Write-up (graded against a likewise designed rubric

Teacher assessed student participation (subjective – based on a likewise designed rubric) EXTENSIONS: ACTIVITIES: none REFERENCES: none

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Student Pages: Formal Lab Write-Up – Day 2 FORMAL LAB WRITE-UP POSE QUESTION (what were you looking to prove, solve, understand etc…)

o What are we trying to do in this investigation? o Due date:

RESEARCH NEEDED TO CONDUCT EXPERIMENT o How did we gather nucleotide sequence, how was the RNAi obtained etc… o What journals, articles, other sources were used to assist in the investigation o Due date:

HYPOTHESIZE – POSE ANSWER, EXPLANATION, OR DESCRIPTION OF OUTCOME o What do you think is going to happen? Will the P25 protein be “silenced”, if so, to what extent? o Due date:

CONDUCT SYSTEMATIC OBSERVATIONS o Provide ongoing data collected throughout the investigation o Keep a journal of each day’s lessons o Due date:

ILLUSTRATE EXPERIMENTAL DESIGN o Provide a digital representation (photo) or illustration (if you have the talent) o Illustrations may include: apparatus set-up and/or silkworm life cycles o Due date:

PROVIDE DETAILED SUMMARY OF INVESTIGATIVE PLAN AND PROCEDURE o List the sequential methods of this investigation from beginning to end. o Include preliminary instructional sessions that gave knowledge/skill for the success of the investigation o Use of dialogue and illustration is desirable o Due date:

1. EXPLAIN METHODOLOGIES TO GATHER INGREDIENTS/COMPONENTS

Within body of summary – include how components were gained (i.e. DNA samples, RNAi targets) Due date:

2. EXPLAIN TOOLS USED TO GATHER DATA

Within the body of the summary include measurement techniques used and what tools Due date:

3. GATHER DATA(MEASUREMENTS IN METRIC)

Include data collection in body of summary (i.e. mass, length, width, cycle times, color etc…) Due date: March 9, 2013

4. GENERATE EXPLANATIONS THAT EXPLICATE OR DESCRIBE WHAT OCCURRED

Include reasoning as to events that occurred during investigation (i.e. contamination of food source caused excessive mold growth etc…)

Due date:

GIVE SUBJECTIVE OPINION OF OVERALL RESULTS – SHARE THIS WITH OTHERS o Explain in your own words/opinions rationale behind outcomes, sources of success/error, overall validity

of hypothesis, means of correction or altering investigation to produce additional results o Share your results with members of other groups investigating, compare outcomes – record at least 2

other interactions of groups. o Due date:

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COMMUNICATE RESULTS OF INVESTIGATION TO SOURCES OTHER THAN INSTRUCTOR o Similar to above – share results with other members of the student or faculty body and record interactive

response o Possible inquiries

How did the recipient respond to the general idea of the investigation? Were they surprised with this type of technology? Did they seem genuinely interested?

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Student Pages: Data Collection Worksheet

DATA COLLECTION WORKSHEET

DAY – DATE GENERAL OBSERVATIONS EXPERIMENTAL

CHANGES RATIONALE FOR

CHANGES

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Appendix #1

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Appendix #2 Exit Card

Lab Group # _________

Which lane demo’s shortest fragment? #____

Lane #5 has which m-stage? ______________

Lane #6 has which m-stage?_______________

Lane #7 has which m-stage?_______________

Did you find the application of RNAi to the leaves

to be challenging? Yes or No (circle)

A Bio-Tech Study in Gene Silencing and RNA Interference

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Transcript of A Bio-Tech Study in Gene Silencing and RNA Interference