Unit 5Cloning

Lesson 1 Introduction to Cloning

Introduction to cloning, gene cloning, DNA libraries. - Lecture and respond to questions.

1 A -Introduction to cloning

• Cloning – A procedure to produce multiple identical copies of a biological unit such as a gene, a cell, or an organism.

Introduction to cloning There are 3 types of cloning:• Gene cloning produces copies of

genes or segments of DNA.• Reproductive cloning produces

copies of whole animals or plants. • Therapeutic cloning produces

embryonic stem cells for experiments aimed at creating tissues to replace injured or diseased tissues.

Gene Cloning• Use restriction enzymes to cut

DNA of interest and cut plasmid.• Ligate DNA of interest and

plasmid.• Introduce recombinant

plasmid to new organism via transformation.• Introduction of foreign DNA

changes (transforms) properties of the organism.

Gene Cloning• After transformation, cells are plated onto agar medium that

contains selective antibiotic: only transformed cells, that acquire antibiotic resistance gene on the vector plasmid, will survive and form colonies.

DNA Libraries Did you ever wonder

how you go about finding DNA or a gene of interest to clone?• If you wish to clone a

gene, either for study or to make a protein product, you have to use methods to isolate this gene from all the other genes in the organism of interest.

DNA Libraries

• One approach to cloning a gene is to construct a DNA Library.• A DNA library is a collection of DNA fragments that have been cloned

into vectors so that researchers can identify and isolate the DNA fragments that interest them for further study.• There are 2 types of DNA Libraries1. Genomic Library2. cDNA Library

DNA Libraries• Genomic Library• Genomic library contains DNA

fragments that represent the entire genome of an organism.• DNA is isolated from an organism.• DNA from the organism is cut with

the same restriction enzyme as the vector plasmid. • Genomic fragments and vector

plasmid are mixed with DNA ligase..• Recombinant DNA is formed.

DNA Library• Genomic Library• Recombinant DNA is inserted into

E.coli.• One plasmid( one DNA fragment) is

inserted into one cell. • Can plate and grow bacterial cells;

each colony has one different DNA fragment. • Several clones are needed to

represent the entire genome.• Can then store organisms. • http://www.sumanasinc.com/webco

ntent/animations/content/dnalibrary.html

DNA Libraries• cDNA Library• cDNA library is a library of actively

expressed genes. There are no introns or non-coding sections of DNA•mRNA is isolated from a tissue of

interest.•mRNA cannot be cut directly with

restriction enzymes.• Reverse transcriptase is used to catalyze

a complimentary DNA strand (cDNA).•mRNA is degraded by enzymes.

DNA Libraries• cDNA Library• DNA polymerase use to construct

second DNA strand.• DNA linkers (restriction sites) are

added to the DNA strands so they can bind to the vector.• DNA strand is mixed with a vector;

most often a plasmid. • Plasmids are transferred to

bacterial cells as with genomic libraries. • http://

highered.mheducation.com/sites/0072556781/student_view0/chapter14/animation_quiz_3.html

DNA Libraries• Genomic vs. cDNA• Genomic libraries are preferred if a biotechnologist’s interest are

entire genomes.• Genomic libraries contain exons and introns for eukaryotic genomes.• __________________________________• cDNA libraries are preferred if the biotechnologist’s interest are

expressed genes because bacteria cannot remove introns from DNA.• _____________________________________• Today, companies manufacture DNA libraries made from different

tissues in a wide variety of organisms.

DNA Libraries• http://www.youtube.com/watch?v=SvjeCxVu2dI• Video genomic and cDNA libraries

DNA Libraries

• Selection or Screening• Now that you have created the library, how do you find the gene that

you want?• Often you know part or all of the DNA sequence of interest or you

may know the DNA sequence for the same gene in a different organism than the one you are studying.• The most common approach to selecting the DNA of interest from a

DNA library is called DNA hybridization (Colony hybridization).• It relies on the fact that a single strand of DNA will hybridize to a

complementary sequence.

DNA Libraries• Colony Hybridization• The bacteria from the DNA

library are plated onto an agar plate• Each colony that grows is from a

single bacteria with a different DNA fragment. Each different colony = one plasmid type.• Each colony is given a number.• A membrane is placed over the

cells and some cells attach to the membrane.

DNA Libraries• The membranes are treated to

lyse bacterial cells and remove debris.• DNA is denatured into single

strands and is still bound to membrane.• A probe, a complimentary single

strand of DNA, is introduced. It is tagged with a radioactive or flourescent dye.• The membrane is incubated and

the probe and DNA of interest bond; called hybridization

DNA Libraries•Membrane is washed to remove

unused excess probe.• Photographic film is used in an

imaging technique called autoradiography.• Anywhere the probe is bound to the

filter, silver grains appear on the film• The film is compared to the original

numbered agar plate and those colonies can be isolated and grown on a larger scale for DNA study.

DNA Libraries• http://sites.sinauer.com/cooper6e/animation0412.html• Screening a DNA library using colony hybridization

Lesson 1A – What you need to know.

• Define cloning.• Describe the 3 types of cloning.• How do you clone a gene?• Define a genomic library.• Explain in detail how you create a genomic library.• Define a cDNA library.• Explain in detail how you create a cDNA library.• Describe in detail the procedure for colony hybridization.

Lesson 2 – Gene Cloning Plants

2A – Lecture : Gene Cloning Methods 2 B- Read articles and write synopsis of articles: Plant Applications Presentations of articles and class discussion

Lesson 2A -Gene Cloning- Plants

• GMO, Cisgenic, Transgenic• GMO - Genetically modified organisms (GMOs) are living things

whose DNA has been changed by humans using the techniques of genetic engineering. The mechanism for creating a GMO is called the recombinant DNA technique.• Cisgenic - Cisgenic organisms are GMOs that result from the

combination of different strands of DNA, which all originate from the same, or very similar, species. All of the DNA used in the process comes from organisms that are very closely related, usually defined as organisms that are able to breed together.

Gene cloning - Plants• Transgenic - Transgenic organisms are genetically modified organisms

that include DNA from two different species. Transgenic microorganisms are the most commonly created transgenic organisms. The simple DNA of bacteria lends itself to relatively straightforward recombinant DNA methods.

Gene Cloning - Plants• Methods • Plant cells are different from animal cells

because many types of plants can regenerate from a single cells.• This natural ability of plants to clone makes

them ideal for genetic research.•When new genetic material is introduced to

a plant cell, it rapidly matures into a plant and results of genetic modifications can be seen in a relatively short amount of time

Gene Cloning - Plants• Methods – Protoplast

Infusion• When a plant is injured, a

mass of cells called a callus grows over the site of the wound.• Callus cells can redifferentiate

into specialized cells and grow into a new plant.• You have taken advantage of

this if you have rooted a plant cutting.

Gene Cloning - Plants• Methods – Protoplast Fusion• Callus cells are surrounded by cellulose.• Cellulose can be dissolved with

cellulose.• The denuded cell is called a protoplast.• The protoplast from one species can

be fused with the protoplast from another species to create a hybrid plant.• Examples – broccoflower (cauliflower

and broccoli).

Gene Cloning - Plants• Methods – Leaf Fragment Technique• Create a recombinant plasmid using the Ti plasmid from

Agrobacterium tumefaciens and a gene of interest (like we read about in the golden rice webquest). http://media.pearsoncmg.com/bc/bc_russell_igen_mol_3/animations/ch17/ch17_a3.swf• Cut a small disc of leaf tissue from a leaf.• When the leaf starts to regenerate into a plant, put it in a

medium with the recombinant A. tumefaciens and the DNA is transferred to the plant genome.• Add plant hormones to regenerate into a new plant.

Gene Cloning - Plants

Gene Cloning - Plants• Methods – Gene Guns• Instead of relying on microbial vehicles for DNA transfer,

researchers can use a gene gun to blast tiny metal beads coated with DNA into an embryonic plant cell.• DNA can be shot into the nucleus or a chloroplast.• The process is hit or miss but transformed cells are identified by

using a marker gene.• The gene of interest is paired with a gene for antibiotic resistance

(marker gene) and plants are grown in a medium with the antibiotic.• Plants that grow have the gene of interest.

Gene Cloning - Plants

Gene Cloning - Plants• Methods – Chloroplast Engineering• Instead of incorporating DNA into the nucleus, DNA of interest can be

introduced into chloroplast DNA.• The DNA (gene) is flanked with chloroplast DNA on each end to ensure it

will be incorporated into the chloroplast.• A microbial vehicle such as the Ti plasmid or gene guns are used to insert

the DNA into the leaf.• Advantages - There are 10 – 100 chloroplasts per cell; greater gene expression . - Chloroplast DNA is inherited through maternal line. Pollen will not have the DNA and cannot be spread to wild type plants.- More than one gene can be inserted into a chloroplast.

Lesson 2A – What you need to know• Compare and contrast the terms GMO, cisgenic, and

transgenic.• Why are plants ideal for genetic research?• Explain protoplast fusion.• Describe in detail how a gene of interest is inserted into a Ti

plasmid.• Explain the leaf fragment technique.• How do gene guns insert DNA into plants?• Explain the method of chloroplast engineering as well as the

advantages of this method.

Lesson 2B – Practical Applications Gene Cloning in Plants• Vaccines for Plants• Crops are vulnerable to a wide range of viruses.• A gene(s) from a weakened virus can be inserted into a Ti plasmid in A.

tumefaciens.• A. tumefaciens infects the plant DNA.• When the plant is exposed to the virus, the inserted virus gene(s)

produce a protein(s) that confers “immunity” to the plant. • Exactly why this works is still being investigated as plants unlike animals

do not have an immune system.• Plant vaccines have revitalized the papaya industry in Hawaii.• • http://www.nytimes.com/1986/05/06/science/plants-vaccinated-against-virus.html

Practical Applications - Plants

• Gene Pesticides• Bacillus thuringiensis (Bt) contains the Cry gene which produces a

crystallized protein.• When the crystallized protein is ingested by certain insects, a toxin is

produced that breaks down the cementing substance that fuses the lining cells of the insect’s digestive tract. The insect dies.• In the past, farmers have spread the crystal over crops but today

plants can be bioengineered to contain the Cry gene.

Practical Applications - Plants• The Cry gene is inserted using the Ti plasmid

of A. tumefaciens.• This Bt toxin has been successfully inserted

in corn, soybeans, cotton, tomatoes, and tobacco.• Controversy: Bt Toxin kills Monarch

butterflies. Research has show that few butterflies are exposed to enough Bt toxin on pollen to cause harm.• http

://www.ext.colostate.edu/pubs/crops/00707.html

Practical applications - Plants

• Herbicide Resistance• Weed killers often kill desirable plants along with the weeds.• Crops have been genetically engineered to be resistant to common

herbicides such as glycophosphate (Roundup).• The herbicide works by blocking the enzyme EPSPS which functions

in a biochemical pathway to produce amino acids vital to plant growth.

Practical Applications - Plants• Scientists have bioengineered a crop

that produces a glycophosphate insensitive EPSPS enzyme.• The altered enzyme is inserted into a

Ti plasmid and the leaf fragmentation method is used to produce the transgenic plant.• The plant is now resistant to

glycophosphate.• Unfortunately, in regions where

glycophosphate resistant trangenics dominate, glycophosphate resistant weed are evolving.

Practical Applications - Plants

• Future engineering of transgenics will have to combat this development.• http://www.nature.com/ne

ws/case-studies-a-hard-look-at-gm-crops-1.12907

Practical Applications - Plants• Enhanced Nutrition• Biotechnology presents the opportunity to save millions of

people from malnutrition.• Improving the nutritional quality of food is being heavily

researched.• The Golden Rice project has genetically engineered rice to

produce beta carotene with the hopes of combatting Vitamin A deficiency.• Also under development are tomato varieties with increase

lycopene content ( a component of Vitamin A) and canola oil with enhanced Vitamin E content. • http

://www.isaaa.org/resources/publications/pocketk/41/default.asp

Practical Applications - Plants• Pharmacology (Biopharming)• Human Vaccines• Plants can produce edible vaccines for humans.• A gene for a subunit of a virus or bacterium

can be inserted into a plant. • The plant expresses the protein and this

antigenic protein enters the blood stream of humans after plant consumption.• Antibody is produced by the human who is

now immune to the pathogen.• http://www.fraunhofer.de/en/press/research-n

ews/2013/june/automated-plant-factory-for-the-prodution-of-vaccines.html

Practical Applications - Plants• Pharmacology (Biopharming)• Hormones and Drugs• Currently most pharmaceuticals are produced by

microorganisms.• In the not too distant future, genes for creating

pharmaceuticals will be inserted into plants.• Insulin and human growth hormone have been produced

using plants.• The FDA has approved a drug to treat Gaucher’s disease.

The drug is manufactured from proteins made from carrot cell cultures.• http://www.ext.colostate.edu/pubs/crops/00307.html

Lesson 2B - What you need to know

• Explain how plant immunity to viruses is conferred.• How are Bt crops made pest resistant?• Explain the method for introducing herbicide resistance in

plants.• How are human vaccines made in plants?• What are the benefits and risks of biopharming?

Lesson 3 Gene Cloning - Animals• Lecture: Methods for gene cloning and practical application• Create a Powerpoint and make a presentation to solicit

funding for your transgenic animal.

Gene Cloning - Animals• Process of introducing new genetic material to organisms is

not limited to plants.• The first experiments in animal transgenics were conducted

at the cellular level.• A new gene was added to an animal cell culture. • Cells were then screened to discover which ones exhibited

the desired gene.• Cells were selected and cloned into a complete animal.

Gene Cloning - Animals• Methods• Retrovirus mediated transgenics• A gene of interest is inserted (in its RNA form) into a

retrovirus.• The retrovirus has a natural affinity to infect cells and

incorporate itself into cellular genomes.• Mouse embryos are infected with the retrovirus vector.• Embryos infected with the virus are selected and implanted

in a surrogate mother.• Baby mice are born with the capacity to produce the

protein from the gene of interest.

Gene Cloning - Animals

• Methods• Pronuclear microinjection• The injection of a DNA solution into pronuclei of fertilized eggs is the

most common method for making transgenic animals.• Injection is done at the stage of development when the ova have two

pronuclei, one from each gamete, which will later fuse to form the diploid nucleus• The construct DNA( gene of interest) is then microinjected into the

pronuclei of each egg. • These eggs are cultured overnight to the 2 cell stage and then

transferred to surrogate mothers .• http://www.youtube.com/watch?v=h-Bfc1GPWpE

Gene Cloning – Animals Embryonic Stem Cell Method

Gene Cloning - Animals• Embryonic Stem Cell Method• Embryonic stem cells are extracted from a

blastocyst.• The DNA of interest is mixed with the stem

cells.• Some of the stem cells will absorb the DNA.• Those stem cells are selected and

microinjected into a host blastocyst.• The host blastocyst is implanted into a

surrogate mother.• http://

www.dnatube.com/video/5561/What-is-Blastocyst-Microinjection

Practical Applications - Animals

http://www.youtube.com/watch?v=_6Q5oM94bOQ

Practical Applications - Animals• Increasing Agricultural Production• Gene transfer has been used to improved productivity in farm

animals.• Eggs have been made healthier by lowering cholesterol content.• Transgenics have improved milk production, increased protein

content in milk, and lowed fat content in milk.• Researchers are studying disease resistant genes for prevention of

foot and mouth disease, cholera, and Newcastle disease in animals.• EnviroPig is a transgenic pig designed to produce 30% phosphorous

in its urine and feces which are major pollutants at pig farms.

Practical Applications - Animals• Scientists are developing antimicrobial genes to insert into farm

animals to prevent food poisoning and reduce antibiotic use in humans.• Researchers are trying to trangenically transform animals into more

efficient grazers, so that more animals can be sustained on the same land.

Practical Applications - Animals• Transgenic Animal Bioreactors• Proteins are important biotechnological

products.• Transgenic animals can be

bioengineered to produce proteins.• Animal bioreactors are engineered as

follows: - The desired gene is introduced into a target cell. - Using cloning techniques, the target cell is raised to an adult animal. - The animal will produce milk or eggs rich in the protein

Gene Cloning - Animals• Biosteel is a product used to

make bulletproof vests and sutures in operating rooms.• Biosteel is make of spider silk,

one of the strongest fibers on earth.• The gene for the silk protein

is inserted into goats.• Goats produce silk milk and

the milk is purified to remove the protein.

Gene Cloning - Animals

• Eggs are another efficient method for producing biotech products.• An average hen produces 250 eggs/year and each egg

generates about 3.5 grams of protein.• Products such lysozyme and virus vaccines are produced in

eggs.

Gene Cloning - Animals

• Human Monoclonal Antibodies• When a foreign antigen (any

substance that causes antibody production- viruses, bacteria) is introduced into a body, the body produces antibody to that antigen.• The antibody binds to the

antigen and destroys the foreign invader.

Gene Cloning - Animals•Monoclonal antibodies (mAb or moAb) are antibodies that are the

same because they are made by identical immune cellsthat are all clones of a unique parent cell.• To make monoclonal antibodies: - The antigen is introduced into a mouse that makes antibody. - The antibody producing cells in the mouse spleen are isolated. - Tumor cells are grown in tissue culture. - Antibody producing cells and tumor cells are fused to produce a hybridoma. - Antibofy producing hybridomas are cloned and the monoclonal antibodies are isolated.

Gene Cloning - Animals

Gene Cloning - Animals• Monoclonal antibodies are used to treat

cancer, heart disease, and transplant rejection.• There is some research that indicates that

monoclonal antibodies may be a cure for AIDS.• Monoclonal antibodies to HCG (human

chorionic gonadotropin) are used to make pregnancy tests.• Moncolonal antibodies are use to detect

drugs and toxins.

Video: Animal Botechnology

• http://www.youtube.com/watch?v=qCIvAuwaf-o

• 30 minutes

Gene Cloning – Find funding for your transgenic animal• Work in pairs to create a powerpoint and give a presentation

to locate funding for your transgenic animal.• See your handout for details and rubric.

Lesson 3 – What you need to know• How is a retrovirus vector used to clone a gene?• Describe the pronuclear microinjection method to clone a

gene?• How does the embryonic stem cell method work to clone a

gene?• Give 3 examples of improvements in agricultural production

due to gene cloning.• Explain how animals are engineered into bioreactors.• Give 2 examples of proteins produced by animal bioreactors.• How are monoclonal antibodies made?• Give 2 examples of monoclonal antibody applications.

Lesson 4 Reproductive Cloning- Plants• Brief explanation of plant cloning • Video – plant tissue culture• Plant Tissue Culture Lab

Reproductive Cloning- Plants• Tissue Culture• Depending on the type of plant, parts of a

plant, such as cuttings from the leaf, stem, roots, or buds may be used to create new plants.• Plants have cells in the above parts called

meristem cells. Meristem cells are the growing regions of plants and these cells can differentiate into any type of plant cell.• In biotechnology, tissue culture takes

advantage of a plant’s innate ability to regenerate a whole new plant from meristem cells.

Reproductive Cloning- Plants• Tissue Culture• If you take a leaf cutting, called an explant, treat it with

ethanol and bleach, and place it in a sterile Petri dish with media composed of sterile minerals, nutrients, and plant hormones, shoots and roots will begin to grow.• Eventually the plantlets are transferred to soil where they

continue to grow into a new plant.• These new plants are disease free because they were

planted in sterile conditions and each leaf section produces a clone of the original plant.

Reproductive Cloning- Plants

http://www.youtube.com/watch?v=__TXXoxcCTI

Reproductive Cloning- Plants• Read pre-lab information• Day 1 : Conduct lab• It will take several weeks for the leaf fragment to mature

into a new plant. We will periodically check the growth and health of our clones.

Lesson 4 – What you need to know.

• Describe the procedure for plant tissue culture.• You will be responsible for questions asked on the lab

handout.

Lesson 5 Reproductive cloning– Animals

• Read Powerpoint about animal cloning procedure and limits of cloning.• Complete cloning webquest. Respond to all questions• Complete activity: Ethics of Human Cloning

Reproductive Cloning- Animals• Animal Cloning Procedure - Enucleation• An ova (egg) is donated from the egg donor animal.• The nucleus is removed from the egg and the

cytoplasm remains.• A somatic cell with the desired genetic material is

donated by the somatic donor animal.• The somatic cells nucleus is inserted into the ova.• An electrical charge fuses the somatic nucleus with the

ova cytoplasm. • The cell behaves as an embryo and begins dividing.

Reproductive Cloning- Animals• Incubate the egg until it reaches the blastocyst stage.• Implant the blastocyst in a surrogate mother who will give birth to

the cloned animal.

Reproductive Cloning- Animals• Limits to Cloning• The donor cell must come from a living organism. The

possibility of finding an entire genome in an extinct organism is remote. No, we are not able to clone dinosaurs!• Clones are genetic duplicates but they are not exactly

identical because they are shaped by their experiences and environment.• The success rate for animal cloning is low. To create Dolly

the first cloned animal, there were 277 attempts. Only 29 embryos lived longer than 6 days and Dolly was the only embryo to produce a sheep.

Reproductive Cloning- Animals• Many clones are born with defects including enlarged

tongues, kidney problems, intestinal blockage, diabetes, and crippling disabilities due to short tendons.• Clones become old before their time (shorter life span). At

the end of each chromosome are repetitive DNA sequences called telomeres. Telomeres function to protect the chromosome from deterioration. Every time a cell divides, the telomeres get shorter. This limits a cell to a fixed number of divisions and thus determines the life of the cell.• The telomeres in clones are the same length as the somatic

donors. Clones’ cells are older than their biological years.

Webquest - Cloning• Visit the website below:• http://teach.genetics.utah.edu/content/tech/cloning/webqu

est.html• Respond to all questions on your handout• We will review responses.

Activity – Bioethics of Human Cloning• http://

teach.genetics.utah.edu/content/tech/cloning/bioethics.html•We will engage in discussion and try to make decisions about

the ethics of human cloning.• See you handout for directions.

Lesson 5 – What you need to know

• Explain the enucleation process.• What are the limits to cloning?• You will be responsible for the information on the

webquest.

Lesson 6 - Therapeutic Cloning• Group work – Stem Cells• The topic of stem cells has been addressed in introductory biology .

This lesson is a review and refresher for previously learned content.• Work in “expert” groups of 4 to review the powerpoint on

assigned section of content. Develop review questions for content.• Teacher will approve all review material.• Reassign one “expert” from each group assignment to a new

grouping.• New group will review powerpoint together, discuss content and

review questions.•We will review each section in class discussion

Lesson 6 – Therapeutic Cloning

• Activity - Webquest stem cells• Activity- Create a stem cell line• Activity – Leukemia webquest• Activity – Animation: Stem cells in heart repair• Video: Stem Cells Miracle Cure

Therapeutic Cloning• Totipotent Stem Cells• These are the most versatile of the stem cell

types. When a sperm cell and an egg cell unite, they form a one-celled fertilized egg. • This cell is totipotent, meaning it has the

potential to give rise to any and all human cells, such as brain, liver, blood or heart cells. It can even give rise to an entire functional organism. The first few cell divisions in embryonic development produce more totipotent cells.

Therapeutic Cloning

• Pluripotent Stem Cells (Embryonic Stem Cells)• These cells are like totipotent stem cells in that they can give rise to

all tissue types. Unlike totipotent stem cells, however, they cannot give rise to an entire organism.• On the fourth day of development, the embryo forms into two

layers, an an outer layer which will become the placenta, and an inner mass which will form the tissues of the developing human body. These inner cells, though they can form nearly any human tissue, cannot do so without the outer layer; so are not totipotent, but pluripotent. As these pluripotent stem cells continue to divide, they begin to specialize further.

Therapeutic Cloning• Multipotent Stem Cells• These are less plastic and more

differentiated stem cells. They give rise to a limited range of cells within a tissue type. The offspring of the pluripotent cells become the progenitors of such cell lines as blood cells, skin cells and nerve cells.• At this stage, they are multipotent. They

can become one of several types of cells within a given organ. For example, multipotent blood stem cells can develop into red blood cells, white blood cells or platelets.

Therapeutic Cloning•Adult Stem Cells•An adult stem cell is a multipotent

stem cell in adult humans that is used to replace cells that have died or lost function. It is an undifferentiated cell present in differentiated tissue. It renews itself and can specialize to yield all cell types present in the tissue from which it originated.

Therapeutic Cloning

• Induced Pluripotent Stem Cells (IPSCs)• IPSCs are differentiated cells that

have been reprogrammed back to pluripotent stem cells.• The introduction of 4 genes OCT3/4,

SOX2, c-MYC, and KLF4 by a retrovirus into cells reprograms the cells into an earlier stage of differentiation similar to embryonic stem cells.• These genes encode transcription

factors involved in cell development.

Therapeutic Cloning

• IPSCs• IPSCs can be used for patient specific therapies without the

risk of cell rejection.• Cells could be taken from a patient, reprogrammed into an

IPC, and then differentiated into a cell that could combat disease in the patient.• There would be no need for embryonic stem cells.

Therapeutic Cloning• IPSCs• Scientists still do not fully understand how to control

induced pluripotent stem cells.1. They do not understand the degree of pluripotency in

these cells.2. Producing them is inefficient. 1 in 1000 cells exposed

to a reprogramming approach becomes an IPSC.3. The cells require constant feeding in cell culture.4. The cells have low viability after they have been

frozen for storage.5. The cells are prone to forming tumors.6. Occasionally, IPSCs spontaneously revert to

differentiated cells.

Therapeutic CloningActivity – Stem Cell Webquest • Visit the following website and respond to questions in your

handout.• http://stemcells.nih.gov/info/basics/pages/basics1.aspx

Therapeutic CloningActivity : Create a Stem Cell Line• Visit the following website:• http://www.edheads.org/activities/stem1/index.shtml

• Respond to questions in your handout • http://www.edheads.org/activities/stem1/swf/vis/Key_to_Id

entifying_Stem_Cell_Types.pdf

Therapeutic Cloning-Practical Application

• Stem Cell Therapies• Potential and promise are two words frequently used to

describe stem cell therapies. The use of stem cells for treating diseases is largely unproven• The most promising application to date has been for

leukemia.1. Patients receive chemotherapy or radiation to destroy

cancerous white blood cells.2. Patients receive WBC stem cells which proliferate to

normal cells.

Therapeutic Cloning – Practical Applications• The use of stem cells has been successful

providing red blood cells for people with sickle cell disease.• Researchers have injected stem cells from

different sources into damaged heart tissue of mice and pigs (heart tissue does not repair itself well). The stem cells developed into cardiac muscle and improved heart function by 35%. • There have been a few human clinical trials in

which stem cells improved cardiac function. This work shows promise for human heart attack victims.

Therapeutic Cloning – Practical Applications• Stem Cell Therapy• Researchers have demonstrated that embryonic stem cells

can be differentiated to form neurons in mice to show improvement in spinal cord injuries. The FDA has approved the first clinical trial for the use of embryonic stem cells to treat humans with spinal cord injuries.

Therapeutic Cloning – Practical Applications• Challenges for stem cell therapy1. Controlling differentiation –When stem cells are injected

scientists cannot control the spread of cells to other places in the body nor can they control the differentiation of stem cells into tissues other than those that were intended.

2. Injected human embryonic stem cells tend for form tumors.3. Chromosomal abnormalities – Abnormality in chromosome

number( trisomy) occurs frequently when stem cells differentiate

• The most promising therapy appears to be differentiating the stem cells in vitro and them injecting them.

Therapeutic Cloning Activities

• Leukemia Webquest - Visit the following websites and respond to the webquest questions.• http://www.edheads.org/activities/stem2/• http

://learn.genetics.utah.edu/content/tech/stemcells/sctoday/ • Animation: Stems cell in heart repair - Visit the following

website and complete the exercise for both patients. A short quiz will follow.• http://www.edheads.org/activities/stem3/index.shtml

Therapeutic Cloning - Video

• Stem Cell Miracle Cure• http://www.youtube.com/watch?v=sLepKx5z-r0

Lesson 6 – What you need to know

• Describe pluripotent, totipotent, and adult stem cells.• How do you create IPSCs?• What are the advantages and disadvantages of IPSCs?• Describe what stem cell therapies are showing promise in

the treatment of disease.• You are also responsible for the questions on the stem cell

webquest and the leukemia webquest.