Genotyping%20A%20Transgenic%20Mouse%20INK4A%20Knock-out%20

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Genotyping A Transgenic MouseINK4A Knock-out
INK4A Wild-type

• By Natalie Saldana
• Dr. Taha Merghoub

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

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Hypothesis

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

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Why Mice?

• They are genetically similar to human beings
• Affordable and easy to maintain
• Able to reproduce within 3 weeks
• Short life span
• Small

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Transgenic Mouse
Genotype
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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

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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)

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PCR Procedure
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Observations Results
? Ink4A WT
We were successful in genotyping the mice which
we will use for future experiments.
? Ink4A KO
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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.

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Reference

• http//users.ugent.be/avierstr/principles/pcr.htm
  l
• http//www.accessexcellence.org/RC/VL/GG/transgeni
  c.html
• http//users.rcn.com/jkimball.ma.ultranet/BiologyP
  ages/T/TransgenicAnimals.html
• http//sunsite.berkeley.edu/PCR/whatisPCR.html
• http//www.agrf.org.au/Default.aspx?tabid85
• http//ghr.nlm.nih.gov/ghr/glossary/genotyping
• http//www.identigene.com/SWIMX/Products/mouse_gen
  otyping.asp
• http//en.wikipedia.org/wiki/Mouse
• http//en.wikipedia.org/wiki/Genotyping
• http//www.skincancer.org/melanoma/index.php

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Acknowledgements
THANK YOU!

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