Generation of a RNA probe for in situ hybridization Molly McMahon Kelly Christensen.

Generation of a RNA probe for in situ

hybridization

Molly McMahon

Kelly Christensen

Diabetes•Two types of Diabetes Mellitus

– Type 1 -Pancreas produces no insulin

-Managed solely by insulin injections

– Type 2-Body metabolizes sugar differently due to

insulin resistance-Managed by a healthy lifestyle and insulin

therapy

•23.6 million people have type 1 Diabetes in the United States

Background Hypothesis Methodology Summary

Diabetes•Two types of Diabetes Mellitus

– Type 1 -Pancreas produces no insulin

-Managed solely by insulin injections

– Type 2-Body metabolizes sugar differently due to

insulin resistance-Managed by a healthy lifestyle and insulin

therapy

•23.6 million people have type 1 Diabetes in the United States

Type 1 Diabetes• Occurs when body’s

immune system attacks and destroys beta cells in the pancreas

• Insulin dependent because pancreas does not produce insulin, which is needed to break down glucose

• Blood sugar levels must be taken multiple times a day in order to maintain glucose homeostasis


(Beta) Cells

• In type 1 Diabetes the body recognizes cells in the pancreas as foreign invaders, and destroys them

cells do not regenerate from the auto immune response


Zinc Transporter Isoform 8 (ZnT8)


• Zinc is a heavy metal that is important to various pathways in the human body

• Many different zinc transporters in the body which serve many functions

• ZnT8 is an autoantigen• Role is in insulin secretion and maturation • Proposed cell specificity with zinc transporter 8

Hypothesis

Previous studies demonstrate that antibody binding of

ZnT8 is pancreatic beta cell specific. If in situ hybridization

studies demonstrate is beta cell specific then we can state

that ZnT8 expression and function is pancreatic beta cell

specific.


Research question: Is ZnT8 expression beta cell specific?

Abstract• Type-1 diabetes is an autoimmune disease where the white blood cells destroy the β-

cells in the pancreas, resulting in insulin dependency because glucose can no longer be metabolized. This project’s purpose was to determine if zinc transporter isoform 8 (ZnT8) is β- cell specific and is a possible immunological target in the development of type-1 diabetes mellitus. ZnT8 is a protein specifically related to the secretion and maturation of insulin in the pancreas. If in situ hybridization demonstrates that gene expression is β- cell specific, then we can state that ZnT8 expression and function is pancreatic β- cell specific. Using recombinant DNA technology, we isolated RNA from pancreatic islet cells. From that we used first strand synthesis to create a purified copy DNA (cDNA) strand where we destroyed the RNA. We used polymerase chain reaction (PCR) to exponentially amplify the ZnT8 gene sequence using specific primers. After using gel electrophoresis as a negative control we used transformation (the uptake of genetic material) to insert the ZnT8 fragment into a bioengineered vector. We then cultured our bacterial vector and purified the DNA plasmid as a template for an in situ probe. DNA sequencing verified the correct 145 base-pair ZnT8 fragment then used to generate a fluorescence probe. We expect in situ hybridization to localize the presence of the ZnT8 gene within pancreatic β-cells. If successful, the β-cell and ZnT8 will demonstrate specificity. Determining ZnT8 localization is a step in discovering if this gene is in fact an immunological target for the development of type-1 diabetes.


cDNA 1st strand synthesis

Fluorescence In situ hybridization

RNA Isolation

cDNAPCR

ZnT8 Fragments

Insertion of ZnT8 into vector/

transformation

Clone DNA

DNA isolation and sequencing verification

Clone DNA In situ probe

generation

Hybridization on pancreas slide

OverviewOverview

PCR Template Preparation

• RNA isolation– Took cell lysate and

isolated RNA using a anion exchange column

– Using reverse transcriptase and first strand synthesis create cDNA

• forward 5’-AACTGGACAGCGCATCAAAC-3’, reverse 5’-GCTTCTGTCGAAGTTCTCTGTC-3’


PCR/ Gel Electrophoresis• Use PCR

(polymerase chain reaction) to amplify cDNA, for ZnT8 gene

• Electrophoresis used to evaluate success of the PCR reaction (one bar represents product specificity) and ladder (control)


OverviewOverview



RNA Isolation

cDNAPCR

ZnT8 Fragments


transformation

Clone DNA



generation


OverviewOverview

Transformation of Colonies• Transformation: Uptake of

genetic material (a plasmid DNA molecule) into a bacterial cell, which allows it to be replicated in the cell independently of the chromosome


•After incubation at 37°C for 16

hours, four colonies were picked and

used to seed 4ml cultures containing

liquid LB media with 35ug/ml

kanomycin

OverviewOverview



RNA Isolation

cDNAPCR

ZnT8 Fragments


transformation

Clone DNA



generation


OverviewOverview

Cloning Reaction

• Took ZnT8 fragments (PCR product) and cloned into a bioengineered vector

• ZnT8/vector construct positive cells are propagated on agar plate, then further grown


Mini prep• Purification of super

coiled plasmid DNA from bacterial culture

• cDNA plasmid is then purified to be used as template for in situ probe.


DNA Sequencing/ Analysis• Sanger DNA

sequencing – Dye-terminator

chemistry

• Verification of the probe sequence using BLAST– 145 base

pair sequence


OverviewOverview



RNA Isolation

cDNAPCR

ZnT8 Fragments


transformation

Clone DNA



generation


OverviewOverview

Flouresence in situ Hybridization

• Technique used to localize the presence of specific nucleic acid sequences

• Probe sequence generated from 145 bp coding sequence of ZnT8 gene

ZnT8

localization

(purple)

Cells (pink)

Future

• Generation of in situ probe with fluorescent protein

• If beta cells in pancreas slide glow it would provide evidence that ZnT8 (an immunological target) is beta cell specific and could be a component in diabetic development

• ZnT8 autoantibody testing is used to determine if a patient is susceptible to developing type-1 diabetes. – It is known that if ZnT8

autoantibodies are present, there is a 94% likelihood of the immune system attacking the β -cells.

MergedNuclei

GlucagonZnT9

ZnT8 and cell

hybridization- proves specificity

Summary• In summary, we created a clone

containing the ZnT8 mRNA sequence

• 145 probe sequence was verified

• In situ probe was generated using the clones and hybridization is complete

• This allows us to say that ZnT8 is cell specific


A big thanks to Randall Wong and Carrie Toews and the Barbara

Davis Research Molecular Biology Core Facility

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Generation of a RNA probe for in situ hybridization Molly McMahon Kelly Christensen.

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