G ENOTYPING THE E NTIRE C OLONY OF T RANSGENIC M ICE 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

P OLYMERASE C HAIN R EACTION 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

P URPOSE 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 blade o Lysis buffer o Proteinase K o Iso-amyl alochol o Tubes o Pipettes o Centrifuge o NanoDrop Apparatus o Ethanol o 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 ExtractionPCR Preparation Gel Running o Flask o TAE Buffer o Agarose (tablets) o Microwave o Buffer Chamber o Gel container o Gel slits M ATERIALS

P ROCEDURE OF DNA E XTRACTION 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 μ L of lysis buffer and 3 μ L 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 μ L of Iso-amyl alcohol and invert several times to mix 7. Spin at 13,000 rpm for 10 minutes

P ROCEDURE C ONT. 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

I DENTIFYING T RANSGENIC M ICE 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 Sampleng/μL 260/2980 Sampleng/μL 260/

DNA Extraction Results Sampleng/ μL 260/280 Sample ng/ μL 260/

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 centrifuge 6.Place the tubes in fisher vortex to make sure it mixes 7.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 sec 60 °C – 15 sec 72 °C– 30 sec 72 °C– 7 min Ice – repeats 25x

P ROCEDURE : R UNNING THE G EL 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 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 minutes 6. Visualize / photograph gel using uv lamp

R ESULTS :

C ONCLUSION 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

R EFERENCE Weinberg, Robert A. Biology of Cancer. New York: Garland Science, 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, Print. PCR Applications Protocols for Functional Genomics. New York: Academic, Print. American Cancer Society (2007). Breast Cancer Facts & Figures Retrieved from Atlanta: American Cancer Society, Inc. Website: Fayed, Lisa (2007). Famous Celebrity Breast Cancer Survivors. Retrieved May 14,2007, from About.com: Health’s Disease and Condition. Web site: can.htm can.htm

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

Thank You