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Research Project
As a biology co-op student at the Zacharewski lab I have two main projects. The first utilizes cDNA microarray technology to study global changes in gene expression in rat hepatic tissue treated with estrogen. In the second project I will be working in vitro investigating gene expression changes due to treatment with 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD in the rat hepatoma H4IIE cell line.
In the first project, immature ovariectomized female Sprague Dawley rats were dosed once or once daily for three consecutive days with ethynyl estradiol (EE), an orally active estrogen. Liver tissues were harvested from treated and time-matched vehicle control animals 2, 4, 8, 12, 18, 24 and 72 hours after the initial dosing. I performed total RNA extractions and quantification by spectrophotometry and gel electrophoresis, followed by cDNA probe preparation, purification and hybridization to microarrays. The cDNA probes are labeled with fluorescent dye-labeled nucleotides (Cy3-UTP or Cy5-UTP) via direct incorporation. Treated and control probes will be labeled with either Cy3 or Cy5 as well as a dye-swapped replicate to account for dye biases. These probes are then combined and hybridized to the microarrays.
My second project will include many of the same procedures as mentioned above except in this case I will be looking at H4IIE cells in culture. The H4IIE’s will serve as our in vitro model for evaluating hepatotoxicity induced by AhR ligands, specifically TCDD, one of the most potent toxins known to man. I will be performing experiments to study TCDD-induced global changes in gene expression, in temporal and dose responsive studies.
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