Transcript presentation - Harlem Children Society

Genotyping A Transgenic Mouse
INK4A Knock-out & INK4A Wild-type
By: Natalie Saldana
&
Dr. Taha Merghoub
Introduction
Human Disease
Melanoma - serious form of skin cancer
Mouse Model – used to mimic the human disease
Transgenic Mouse
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 beings
Affordable and easy to
maintain
Able to reproduce within 3
weeks
Short life span
Small
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
DNA Extraction
PCR
Gel Electrophoresis
Taq Polymerase
Agarose
Mouse Tails
Buffer
TAE Buffer
Qiagen DNeasy
MgCl2
Ethidium Bromide
Protocol
Primers
Trays & Combs
dNTPs
Gel Box
DNA
Power Supply
H2O
UV Light
1 Kb Ladder
Loading Dye
Thermal Cycler
(PCR Machine)
PCR Procedure
Observations & Results
 Ink4A WT
We were successful
in genotyping the
mice which we will
use for future
experiments.
 Ink4A KO
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_g
enotyping.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