Download Genotyping of Transgenic Mice Population

GENOTYPING THE ENTIRE COLONY OF
TRANSGENIC MICE
By: Sweta Roy & Whitney Lai
Mentor: Dr. Sumanta Goswami
Location: Yeshiva University
KEY TERMS




Primer – a DNA fragment;
used to start DNA synthesis
Buffer solution - solution
that creates a neutral
environment by resisting
any pH changes
Taq Polymerase – DNA
polymerase that creates
matching nucleotides
based from the DNA
template
Transgenic mice- carries a
foreign gene that has
been inserted into its
genome
WAP & MT

Whey Acidic Protein- a gene that codes for
milk protein in certain mammals.
-a Middle T promoter
-found on chromosome 11
-found in dog, domestic; pig, domestic; rabbit,
European; rat

Middle T (MT) – a gene that causes cancer
POLYMERASE CHAIN REACTION
Polymerase Chain Reaction (PCR) is a process that
amplifies DNA through a series of heating and
cooling. Denature, anneal, and elongation are the
basic steps in Polymerase Chain Reaction.
1. Denature: The DNA separates
into two strands
2. Annealing: Primers are added
and forms hydrogen bond with
template
3. Elongation – Primers begins the
replication process  polymerase
is activated; as polymerase runs
through the strand, the
complementary nucleotides are
created with the use of dNTPs
PURPOSE


Goal: To genotype the entire transgenic mice
population
To identify the mice that has both WAP and
Middle-T which are the genes that we desire;
to identify mice with Middle T
MATERIALS
DNA Extraction
PCR Preparation
o
Razor blade

Tubes
o
Lysis buffer

Pipettes
o
Proteinase K

4 μL of DNA of each subject
o
Iso-amyl alochol

5 μL of water
o
Tubes

1 μL of primer
o
Pipettes

10 μL of immomix
o
Centrifuge

PCR machine
o
NanoDrop Apparatus

Box full of ice
o
Ethanol
o
Incubator
Gel Running
o
Flask
o
TAE Buffer
o
Agarose (tablets)
o
Microwave
o
Buffer Chamber
o
Gel container
o
Gel slits
PROCEDURE OF DNA EXTRACTION
1.
2.
3.
4.
5.
6.
7.
Mark the mice; create a code so
you may be able to identify it
Clip the tails of the mice
Place mouse tail in a tube
containing 400 μL of lysis
buffer and 3 μL of proteinase K
Spin at 13,000 rpm for 5
minutes
Draw out all the liquid; leaving
the pellet in the container
Add supernatant to 400 μL of
Iso-amyl alcohol and invert
several times to mix
Spin at 13,000 rpm for 10
minutes
PROCEDURE CONT.
8. Draw out and discard
supernatant (top layer of the 2
layers formed in the tube)
9. Wash with 1mL of 70% ethanol
10. Spin at 13,000 rpm for 5
minutes
11. Air dry for 15 minutes until all
ethanol is gone
12. Add 200 μL of nuclease free
water and incubate in 55
degrees Celsius bath for 1
hour
13. Measure DNA concentration on
Nanodrop Apparatus
14. Ensure that they have ~50ng/
μL concentration of DNA and
add 1 μL of PCR reaction tube
IDENTIFYING TRANSGENIC MICE
Code:
1.
C6#1
2.
C6#2
3.
C6#3
4.
C6#4
5.
C6#0
6.
C1
7.
C5B#1
8.
C5B#2
9.
C5#1
10. C5#2
11. C5#3
12. C5#4
13. C5#0
14. C2#1
15. C2#0
16. C4B#1
C4B #2
18. C4B#3
19. C4B#0
20. C2
21. C9
22. C4#1
23. C4#0
24. C5D#1
25. C5D#0
26. C12#1
27. C12#2
28. C12#3
29. C12#4
30. C12#0
31. C3#1
32. C3#2
17.
33. C3#0
49. C1B#3
34. C4#1
50.
35. C4C#0
36. C4C#3
C1B#4
51. C1C#1
52. C1C#2
37. C2B#1
38. C2B#2
39. C2B#0
40. C2B#0
41. C14#1
42. C14#2
43. C14#0
44. C14males
#1
45. C14males #2
46. C14males #0
47. C1B#1
48. C1B #2
DNA Extraction Results
Sample
ng/μL
260/2980
Sample
ng/μL
260/280
1
4.71
1.37
16
9.96
1.48
2
6.12
1.44
17
21.58
1.56
3
9.23
1.65
18
14.63
1.69
4
13.07
1.50
19
7.40
1.54
5
12.98
1.33
20
28.47
1.57
6
24.22
1.54
21
11.92
1.42
7
24.22
1.34
22
9.81
1.43
8
22.36
1.27
23
35.53
1.52
9
24.82
1.44
24
16.13
1.47
10
13.25
1.49
25
37.34
1.63
11
41.09
1.57
26
9.16
1.56
12
30.47
1.66
27
9.00
1.48
13
6.29
1.31
28
16.06
1.43
14
4.60
1.65
29
10.44
1.39
15
16.65
1.34
30
20.91
1.63
DNA Extraction Results
Sample
ng/μL
260/280
Sample
ng/μL
260/280
31
19.13
1.27
42
9.80
1.29
32
14.61
1.37
43
32.84
1.60
33
11.20
1.36
44
18.35
1.58
34
12.78
1.65
45
7.80
1.14
35
20.12
1.53
46
41.70
1.61
36
19.96
1.54
47
21.97
1.51
37
4.31
1.33
48
37.62
1.54
38
6.71
1.25
49
57.66
1.61
39
6.57
1.36
50
57.70
1.59
40
14.50
1.59
51
33.10
1.63
41
22.55
1.40
52
19.06
1.68
Procedure: PCR
1.
2.
3.
4.
5.
6.
7.
8.
Add 5 μL water
Add 4 μL of DNA (the average)
Add 10 μL of Immomix
Add 1 μL of primer
n uL DNA + n uL Water = 9 uL
Spin the tubes in the centrifuge
Place the tubes in fisher vortex to make sure it mixes
Spin the tubes in centrifuge again
Place the tubes in the PCR machine and run the Genotype PCR program
Genotype PCR Program
94°C– 7 min (starts the cycle)
95 °C – 15 sec
60 °C – 15 sec
72 °C– 30 sec
72 °C– 7 min
Ice –
repeats 25x
PROCEDURE : RUNNING THE GEL
Making a gel
1. In a 500 mL flask, add 1g of agarose ( 2 tablets of agarose)
2. Add 50 mL of 1xTAE buffer to the flask
3. Heat in microwave for less than 1 minute. Watch until bubbles appear
4. Allow the liquid to cool off a bit, about 2-3 minutes or so.
5. Once its no longer boiling hot, add ethidium bromide to a final
concentration of .5 μL/mL
6. Pour into gel cast and wait for gel to harden, approximately 10-15 mins
7. Pour TAE buffer in the Buffer Chamber
8. Place the hardened gel that is still in the slot in the Buffer chamber; the
buffer should cover the gel slightly
DNA Prep
1. To your amplified DNA sample, add loading dye in appropriate
volume; add 4 μLof 6x Loading Dye
2. Mix DNA and dye well
3. Add about 10 μL DNA to each well
4. In addition to DNA add 3-4 μL DNA ladder to one of the wells
5. Run the gel at around 100 v for 30-40 minutes
6. Visualize / photograph gel using uv lamp
RESULTS:
CONCLUSION

From our results we can see that tube 11and
tube 5 have both middle T and WAP which are
the genes we desire.

The ones that have WAP and Middle T will grow a
tumor within two months
Future

We are going to finish the experiment and see
what results we get.

Then we will breed the mice who does not
have one of the genes we desire.

Try to reduce the WAP population
REFERENCE





Weinberg, Robert A. Biology of Cancer. New York: Garland
Science, 2006. Print.
Grobstein, Ruth H. The Breast Cancer Book What You Need to
Know to Make Informed Decisions (Yale University Press Health
& Wellness). New York: Yale UP, 2005. Print.
PCR Applications Protocols for Functional Genomics. New York:
Academic, 1999. Print.
American Cancer Society (2007). Breast Cancer Facts &
Figures 2007-2008. Retrieved from Atlanta: American Cancer
Society, Inc. Website:
http://www.cancer.org/downloads/STT/BCFF-Final.pdf
Fayed, Lisa (2007). Famous Celebrity Breast Cancer Survivors.
Retrieved May 14,2007, from About.com: Health’s Disease and
Condition. Web site:
http://cancer.about.com/od/celebritytributes/a/famousbreast
can.htm
ACKNOWLEDGEMENTS
Dr. Sumanta Goswami
 Joshua Bernstien
 Robert Stobezki
 Josh Jay
 Yeshiva University
 Mice
 Dr. Sat
 Harlem Children Society
 You

Thank You