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 REACTIONPolymerase 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

o Razor bladeo Lysis buffer o Proteinase K o Iso-amyl alocholo Tubeso Pipettes o Centrifugeo NanoDrop Apparatuso Ethanolo Incubator

Tubes Pipettes 4 μL of DNA of each subject 5 μL of water 1 μL of primer 10 μL of immomix PCR machine Box full of ice

DNA Extraction PCR Preparation

Gel Running

o Flask o TAE Buffero Agarose (tablets)o Microwaveo Buffer Chamber o Gel containero Gel slits

MATERIALS

PROCEDURE OF DNA EXTRACTION

1. Mark the mice; create a code so you may be able to identify it

2. Clip the tails of the mice

3. Place mouse tail in a tube containing 400 μL of lysis buffer and 3 μL of proteinase K

4. Spin at 13,000 rpm for 5 minutes

5. Draw out all the liquid; leaving the pellet in the container

6. Add supernatant to 400 μL of Iso-amyl alcohol and invert several times to mix

7. 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 17. C4B #2 33. C3#0 49. C1B#3

2. C6#2 18. C4B#3 34. C4#1 50. C1B#4

3. C6#3 19. C4B#0 35. C4C#0 51. C1C#1

4. C6#4 20. C2 36. C4C#3 52. C1C#2

5. C6#0 21. C9 37. C2B#1

6. C1 22. C4#1 38. C2B#2

7. C5B#1 23. C4#0 39. C2B#0

8. C5B#2 24. C5D#1 40. C2B#0

9. C5#1 25. C5D#0 41. C14#1

10. C5#2 26. C12#1 42. C14#2

11. C5#3 27. C12#2 43. C14#0

12. C5#4 28. C12#3 44. C14males #1

13. C5#0 29. C12#4 45. C14males #2

14. C2#1 30. C12#0 46. C14males #0

15. C2#0 31. C3#1 47. C1B#1

16. C4B#1 32. C3#2 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. Add 5 μL water 2. Add 4 μL of DNA (the average) 3. Add 10 μL of Immomix 4. Add 1 μL of primer 5. Spin the tubes in the centrifuge6. Place the tubes in fisher vortex to make sure it mixes7. Spin the tubes in centrifuge again 8. Place the tubes in the PCR machine and run the Genotype PCR program

n uL DNA + n uL Water = 9 uL

Genotype PCR Program 94°C– 7 min (starts the cycle)95 °C – 15 sec60 °C – 15 sec72 °C– 30 sec72 °C– 7 min Ice –

repeats 25x

PROCEDURE : RUNNING THE GELMaking 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 flask3. 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 mins7. 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 Prep1. To your amplified DNA sample, add loading dye in appropriate

volume; add 4 μLof 6x Loading Dye2. Mix DNA and dye well3. 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

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

Future

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/famousbreastcan.htm

 

ACKNOWLEDGEMENTS

Dr. Sumanta Goswami Joshua Bernstien Robert Stobezki Josh Jay Yeshiva University Mice Dr. Sat Harlem Children Society You

Thank You