Genotyping A Transgenic MouseINK4A Knock-out & INK4A Wild-type

By: Natalie Saldana

&

Dr. Taha Merghoub

Introduction

Mouse Model – used to mimic the human disease

Melanoma - serious form of skin cancer

Transgenic Mouse

Human Disease

Melanoma A rare type of skin cancer that originates in the

melanocytes - found predominantly in skin but also in the bowel and the eye

Causes: Sun exposure - UVA and UVB rays are can

induce skin cancer Moles - normal moles — the small brown

blemishes, growths, or "beauty marks" atypical moles -- also known as dysplastic nevi

Skin Type - fairer skin are at increased risk Family History Personal History - Once you have had melanoma,

you run an increased chance of recurrence Weakened Immune System - immune systems

effected with chemotherapy, an organ transplant, excessive sun exposure, and diseases such as HIV/AIDS or lymphoma can increase your risk of melanoma

Hypothesis

The introduction of Human melanoma mutation in mice will induce this disease in mice

Why Mice?

They are genetically similar to human beingsAffordable and easy to maintainAble to reproduce within 3 weeksShort life spanSmall

Transgenic Mouse

Genotyping

The process of testing individuals in order to reveal any specific gene that has been inherited from that individual

Includes other methods like PCR, DNA sequencing, and Southern blot

The goal is to understand the functions and roles of genes and to test new therapies in melanoma

Materials PCR

Taq Polymerase

Buffer

MgCl2

Primers

dNTPs

DNA

H2O

Gel Electrophoresis

Agarose

TAE Buffer

Ethidium Bromide

Trays & Combs

Gel Box

Power Supply

UV Light

1 Kb Ladder

Loading Dye

DNA Extraction

Mouse Tails

Qiagen DNeasy Protocol

Thermal Cycler (PCR Machine)

PCR Procedure

Observations & Results

Ink4A WT

Ink4A KO

We were successful in genotyping the mice which we will use for future experiments.

Conclusion and Future Directions

We have identified Heterozygous and KO Ink4a mice.

The Ink4a mice will be crossed with NRas transgenic mice in order to obtain a mouse with melanoma.

Reference

http://users.ugent.be/~avierstr/principles/pcr.html http://www.accessexcellence.org/RC/VL/GG/

transgenic.html http://users.rcn.com/jkimball.ma.ultranet/

BiologyPages/T/TransgenicAnimals.html http://sunsite.berkeley.edu/PCR/whatisPCR.html http://www.agrf.org.au/Default.aspx?tabid=85 http://ghr.nlm.nih.gov/ghr/glossary/genotyping http://www.identigene.com/SWIMX/Products/

mouse_genotyping.asp http://en.wikipedia.org/wiki/Mouse http://en.wikipedia.org/wiki/Genotyping http://www.skincancer.org/melanoma/index.php

Acknowledgements

Dr. Taha Merghoub Diana Hong Houghton Laboratory Dr. Sat Bhattacharya Harlem Children Society