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First Work Term:
As an undergraduate co-operative education student I was asked
to participate in the toxicogenomics project under the direction
of Dr. Halgren. Having arrived near the beginning of the project
I used public databases from NCBI's Unigene, Jackson Laboratories,
GeneCards and GenAtlas to search for functions of genes expressed
in mouse testis. To narrow down the options of genes to be place
on the gene chip, human orthologues are being found through
the use of NCBI's BLAST program. My laboratory experience requires
me to oversee the handling of IMAGE clones ordered for the purpose
of the gene chip. All clones received must be sequence verified
before use. For cost effectiveness of sequencing, DNA from all
clones are run on agarose gels for qualitative analysis. By
prescreening, we can determine which clones will require additional
attention to assure clean DNA is delivered to the sequencing
facility.
Other experience which I have gained from Dr. Zacharewski's
lab is handling, dosing and dissection of mouse and Xenopus
species. Upon my return to the lab in May, I hope to continue
research with the DNA chip being produced.
June 5 University of Waterloo Daily
Bulletin article about Cora's First Work Term.
Second Work Term:
For my second semester as a co-operative education student
in Dr. Zacharewski's lab I am continuing to work on the microarray
project. As the sequence verified clones are now being used
for cDNA chip printing, the next phase of the project is to
optimize protocols for the hybridization of mRNA extracts from
mouse testis samples.
For microarray experiments, fifty micrograms of mRNA is required
to obtain a readable signal, with fluorescent Cy3- or Cy5-dUTPs
dyes. The amount of mRNA which can be extracted from tissues
under investigation is much less than this minimum requirement.
Therefore an amplification proceedure is necessary.
Working under the supervision of Mark Fielden, I am testing
a protocol to linearly amplify mRNA. Whereas PCR experiments
will increase DNA concentration exponentially because of a double
stranded template, mRNA is only increased in a linear fashion
due to its single stranded nature. Linearly amplified RNA should
be indicative of all genes expressed in the tissue at any one
time point. If the protocol is successful, mRNA collected from
single cells or smaller tissue samples can also be used in future
hybridization experiments.
It is expected, by end of June, that hybridization experiments
will begin using samples collected in the previous year.
Other projects on the side include more animal handling work
with mice such as dissecting mammary glands for whole mount
staining and collecting other tissues samples for RNA extraction.
Update August 23, 2000
For the linear amplification project, all conditions for the
in vitro transcription have been optimized to present a yield
of approximately 10mg of amplified product from 1mg of total
RNA. Unexpectedly, the fluorescent labeling of the amplified
product has displayed low signal. Experiments involving the
linearity or non-linearity of the amplified product will be
postponed until a more efficient labeling protocol can be found.
In the event that the RNA amplification is not linear, we are
hoping that the experimental variance of the technique itself
will be minimal allowing two amplified samples to be compared.
Regarding the mouse DES study under Mark Fielden, RNA has been
extracted from all male testes and mammary gland whole mounts
have been structurally analyzed.
Prior to my second departure I helped to continue the estrogen
induced vitellogenin expression in Japanese male quail project
established by Dr. Scott Kramer. By using the TaqMan assay,
vitellogenin mRNA levels are to be determined using fluorescent
probes. Template, primer and probe volumes have been optimized
and Kirsten Fertuck will conduct further experiments.
Third Work Term:
For my third semester in the Zacharewski lab, I have been assigned
several projects.
The BIACore system is being used to study the binding kinetics
of coactivator recruitment by nuclear receptors. It has been
well documented that coactivators are recruited by nuclear receptors,
upon ligand binding, and is a fundamental component of the complexes
formed for transcriptional activation. One coactivator from
each of the three known families, and a corepressor are currently
being cloned for expression and purification. These protein
products will then be subjected to known concentrations of different
receptors and corresponding ligands to determine Kd values for
liganded receptor-coactivator/corepressor interactions.
As a side task, I am running experiments using the TaqMan quantitative
PCR technique. Quantitative PCR is a useful tool to observe
changes in gene expression when an organism is exposed to biologically
exogenous compounds. These experiments are performed to determine
how estrogenic chemicals have affected vitellogenin mRNA levels
in male Japanese quail livers. Vitellogenin is a biomarker known
to be induced by estrogenic compounds and is found in egg producing
animals. Only male animals are studied because they do not generate
high levels of vitellogenin and thus give a low background on
such quantitative experiments.
An alternative method to observe gene expression changes, induced
by exogenous chemicals, is through comparison studies on microarrays.
My last two work terms involved working with Mark Fielden by
processing testis samples diethylstilbestrol and genistein treated
mice for use on in house-produced microarrays. In the case of
female animals, mammary gland whole mounts are analyzed for
size, growth, number of terminal end buds and density of differentiating
alveolar buds. Work on this project is also being continued
for this work-semester.
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