Download Research Paper Genotyping the Entire Colony of Transgenic Mice

Genotyping of Transgenic Mice
Genotyping the Entire Colony of Transgenic Mice
Whitney Lai
Yeshiva University
Professor Sumanta Goswami
August 29th, 2009
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Genotyping of Transgenic Mice
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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.
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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
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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.
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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
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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.
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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:
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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:
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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
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