1. Genotyping A Transgenic Mouse INK4A Knock-out & INK4A Wild-type By: Natalie Saldana & Dr. Taha Merghoub

2. Introduction Mouse Model – used to mimic the human disease Melanoma - serious form of skin cancer Transgenic Mouse Human Disease

3. 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

4. Hypothesis  The introduction of Human melanoma mutation in mice will induce this disease in mice

5. Why Mice?  They are genetically similar to human beings  Affordable and easy to maintain  Able to reproduce within 3 weeks  Short life span  Small

6. Transgenic Mouse

7. 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

8. 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)

9. PCR Procedure

10. Observations & Results  Ink4A WT  Ink4A KO We were successful in genotyping the mice which we will use for future experiments.

11. 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.

12. 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_g enotyping.asp  http://en.wikipedia.org/wiki/Mouse  http://en.wikipedia.org/wiki/Genotyping  http://www.skincancer.org/melanoma/index.php

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