Genotyping the Entire Colony of Transgenic Mice

1. Genotyping the Entire Colony of Transgenic Mice By: Sweta Roy & Whitney Lai Mentor: Dr. Sumanta Goswami Location: Yeshiva University

2. 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

3. 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

4. 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.

5. 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

6. 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

7. Materials • Razor blade • Lysis buffer • Proteinase K • Iso-amyl alochol • Tubes • Pipettes • Centrifuge • NanoDrop Apparatus • Ethanol • 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 • Flask • TAE Buffer • Agarose (tablets) • Microwave • Buffer Chamber • Gel container • Gel slits

8. Procedure of DNA Extraction • 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

9. 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

10. Identifying Transgenic Mice Code: • C6#1 17.C4B #2 33. C3#0 49. C1B#3 • C6#2 18.C4B#3 34. C4#1 50. C1B#4 • C6#3 19. C4B#0 35. C4C#0 51. C1C#1 • C6#4 20. C2 36. C4C#3 52. C1C#2 • C6#0 21. C9 37. C2B#1 • C1 22. C4#1 38. C2B#2 • C5B#1 23. C4#0 39. C2B#0 • C5B#2 24. C5D#1 40. C2B#0 • C5#1 25. C5D#0 41. C14#1 • C5#2 26. C12#1 42. C14#2 • C5#3 27. C12#2 43. C14#0 • C5#4 28. C12#3 44. C14males #1 • C5#0 29. C12#4 45. C14males #2 • C2#1 30. C12#0 46. C14males #0 • C2#0 31. C3#1 47. C1B#1 • C4B#1 32. C3#2 48. C1B #2

11. DNA Extraction Results

12. DNA Extraction Results

13. Procedure: PCR • Add 5μL water • Add 4 μL of DNA (the average) • Add 10 μL of Immomix • Add 1 μL of primer • 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 n uL DNA + nuL Water = 9 uL 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

14. 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

15. Results:

16. 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

17. 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

18. 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

19. Acknowledgements • Dr. SumantaGoswami • Joshua Bernstien • Robert Stobezki • Josh Jay • Yeshiva University • Mice • Dr. Sat • Harlem Children Society • You

20. Thank You