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Genotyping of Transgenic Mice Genotyping the Entire Colony of Transgenic Mice Whitney Lai Yeshiva University Professor Sumanta Goswami August 29th, 2009 I Genotyping of Transgenic Mice II Abstract At the Yeshiva University laboratory a group of mice who carries tumors are required for experimentation. However, there are many mice that show no previous records of observation. And so, the paper depicts the genotyping process in identifying the mice before any further research. There are 57 mice in the laboratory; many of those mice are unusable and therefore are wasting the resources and taking up space. By observing their genes we can eliminate unnecessary subjects. To do so, identification of mice that has both WAP and Middle-T, or has only Middle T is important. Mice that are both WAP and Middle T positive will form tumors in approximately two months. However, if the results show that the mice have only Middle T or only WAP, breeding will be performed to produce offspring that have potential in developing tumors. Once the potentially capable of producing tumors mice have been identified, needle injections and other operations would be performed upon them. Genotyping of Transgenic Mice III Genotyping the Entire Colony of Transgenic Mice Breast cancer is one of the challenges that pulverize the self-confidence and self-esteem of millions of women every year. However, there is proof that there is a solution to the hardest of problems. Kate Jackson is a prime example of a woman who has climbed this tower of peril, and came out on the other side. Kate Jackson is a well-known actress who played “Charles Angles.” She has survived from breast cancer two times; once in 1987 and another in 1989 (Fayed, 2007). Her bravery and courage was recognized and her survival from breast cancer gave hope for other breast cancer patients. Although incidence rates have increased by 0.5% per year between 1975 and 2001 (American Cancer Society, 2007), incidence rates have decreased by 3.5% between 2001 and 2004 (American Cancer Society, 2007). To reduce the rising incidence rates even further, Yeshiva University is conducting breast cancer research. There are numerous reasons that cause cancer to develop and the gene Middle T is one of them. WAP is an abbreviation for Whey Acidic Protein; it is a protein that codes for milk proteins in certain mammals such as domestic dogs, pigs, European rabbits, rats, etc. WAP is also a Middle T promoter; meaning, when WAP and Middle T is both present, WAP triggers Middle T causing breast cancer in the subject. In this research, DNA extraction, Polymerase Chain Reaction, and Electrophoresis are performed. The first experiment that is to be performed is DNA extraction; DNA is extracted from each mice. To do so the following materials are required: a razor blade, Lysis Buffer, Proteinase K, Iso-amyl alcohol, Tubes, Pipettes, Centrifuge, NanoDrop Apparatus, Ethanol, and an Incubator. First, mark each mouse and create a code. When conducting an experiment, it is very important to label everything. In this experiment, each mouse in a cage has their tail striped with Genotyping of Transgenic Mice IV a specific number of stripes and was recorded on paper for later analysis. The following is the code that was used: The first number is the number assigned to represent the mouse; it is used for labeling the tubes. C and the number that follows represent the cage number and the number that follows indicates the number of stripes on the mouse. Secondly, using the razor blade, clip a small portion of the mouse tail and place it in a tube containing 400uL lysis buffer and 3uL proteinase K solution; and incubate it in 55 degrees Celsius for 24 hours. The lysis buffer will break down the tail into molecular components while the proteinase K will degrade the proteins in the solution. Then, spin the tube at 13,000 rpm in the centrifuge for five minutes. There should be a pellet at the bottom of the tube in a liquid solution; draw out the liquid and dispense it in a separate tube. The liquid that is just drawn out is a mixture of DNA and other materials such as proteins and lipids. The purpose of this experiment in DNA extraction is to obtain the purest DNA possible. To do so, all the unnecessary components must be removed. Next, add supernatant to 400 μL of Iso-amyl alcohol and invert several times to mix. Afterwards, spin at 13,000 rpm in the centrifuge for 10 minutes. The solution would be separated into two layers. Genotyping of Transgenic Mice V The top layer consists of Iso-Amyl Alcohol and other unnecessary components, while the bottom layer consists of mainly DNA. The purpose of adding Iso-Amyl Alcohol is to aid in the separation of the other components from the DNA. Draw out the top layer and discard it, leaving the rest of the solution in the tube. Remember, to label each tube while doing so. Then, add 1mL of 70% ethanol and spin at 13,000 rpm for 5 minutes in the centrifuge. Open the covers of the tubes and air dry for 15 minutes until all of the ethanol is gone. Next, add 200uL L of nuclease free water and incubate in 55 degrees Celsius bath for 1 hour. An incubator is a machine that controls the atmosphere including the weather, temperature, and humidity of the environment. Finally, measure the DNA concentration on the NanoApparatus, a NanoApparatus, is a machine that measures the quality and concentration of nucleic acids with a drop of the solution. The next experiment that is to be carried out after DNA extraction is 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. The first stage that the DNA goes through is denaturing. The DNA separates into two separate strands called templates. The next stage is annealing; the primers are added to form hydrogen bonds with the template. The final stage that the DNA goes through is elongation; the primer that was added during the previous stage activates the polymerase. As the polymerase runs down the template, it forms complementary nucleotides with Deoxynucleoside triphosphate. Deoxynucleoside triphosphate, also known as dNTP, is a nucleotide with three phosphates. When dNTP bonds with the corresponding DNA strand, the two phosphates dissociates. dNTP serves as “free nucleotides” that is needed to assemble a DNA strand. The materials required for Polymerase Chain Reaction are: Tubes, Pipettes, 4 μL of DNA of each subject, 5 μL of water, 1 μL of primer, 10 μL of Immomix, PCR machine, and a Box full Genotyping of Transgenic Mice VI of ice. After gathering the materials, first add 5 μL water to a tube. Remember to label the tube accordingly to the DNA you are using. Secondly, with the same tube add 4 μL of DNA, 10 μL of Immomix and add 1 μL of Middle T primer. Next, spin the tubes in the centrifuge. Then, place the tubes in fisher vortex to make sure it mixes and spin the tubes in centrifuge again. Finally, place the tubes in the PCR machine and run the Genotype PCR program. Repeat the procedures for all the tubes except, with WAP primer. At the end of the experiment, there should be two sets of amplified DNA: 57 tubes with WAP primer and 57 tubes with Middle T primer. The final experiment that is to be carried out is Electrophoresis which in other words is Gel Running. This process can be broken up into two parts: making a gel, and DNA Preparation. The materials required for Gel Running includes: Flask, TAE Buffer, Agarose (tablets), Microwave, Buffer Chamber, Gel container, Gel cast, Gel slits. First, make a gel by adding 100mL of 1xTAE buffer and 4 tablets of agarose in a flask. Wait until the tablets have dissolved into the solution. Next, heat the flask in a microwave less than 75 seconds; once bubbles appear in the flask, remove the flask from the microwave. The purpose of heating the flask is to make the solution homogeneous. Allow the liquid to cool off. Once its no longer boiling hot, add ethidium bromide to a final concentration of .5 μL/mL. Afterwards, poor the liquid in the gel cast. Wait until the gel to have hardened, about 10-15 minutes. Then pour TAE buffer in the Buffer Chamber and place the hardened gel that is still in the slot in the Buffer chamber; the buffer should cover the gel slightly. After the gel has been made, add loading dye in appropriate volume to the amplified DNA with WAP primer; add 4 μL of 6x Loading Dye. Then, mix DNA and dye well and add about 10 μL DNA to each well. In addition to DNA add 3-4 μL DNA ladder to one of the wells. The DNA ladder serves as the control of the experiment. Afterwards, run the gel at around 100 v for 30-40 minutes. Finally, Visualize / photograph gel using uv lamp. Genotyping of Transgenic Mice VII Repeat the process for DNA samples with Middle T primer and add Meth-1 Middle T to one of the wells; it serves as the control for Middle T primer. The results from the DNA Extraction are shown below: Genotyping of Transgenic Mice VIII The first column labeled “Sample,” contains the identification number that is used to identify the mice. The second column, “ng/uL,” allows us to see if the DNA is usable; in order for the PCR process to work there needs to be a final concentration of 50ng/mL. In the PCR process, the water and DNA amounts needs to add up to 9uL, therefore the approximate minimum amount of ng/mL required for the sample to function properly is 5ng/mL. (50 divide by 9 is 5.56) If the results from the NanoApparatus read to be less than 5, then the sample could not be used. Therefore, the result should be as close to 5 as possible in order for the next process to be taken place (Polymerase Chain Reaction). The third column, “260/280,” measures the quality and purity of the DNA. If the number is less than 2, then the sample is far from being pure; it contains too much unnecessary components such as proteins and lipids. However, if the result says it is greater than 2, then the DNA sample is degrading. Therefore, the result should be as close to 2 as possible in order for the next process to be taken place (Polymerase Chain Reaction). The following are the final results from Gel Running: Genotyping of Transgenic Mice IX The results are incomplete, however our findings show that mice #1,2,3,4,5,6,7,8,9,10,11, and 12, are WAP positive and mice #5 and 11 are Middle T positive. The bars that are circled on the left indicate that the gene for WAP are present, while the bars on the right that are cricled indicates that the gene for Middle T are present. Therefore, mice #5 and #11 will develop tumors in approximately 2 months. Partial elimination of the WAP positive mice will be taken place to reduce the colony size, and breeding of WAP and Middle T positive will be carried out. Although, it may seem that this research is small and insignificant it actually contributes to future discovery and projects. The significance of this research is to provide testable subjects for future research. Genotyping of Transgenic Mice Works Cited 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 X