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Research Project
Estrogenic compounds are known to elicit a myriad of genomic effects via the estrogen receptor. These effects are well-defined within classic target tissues such as the uterus or mammary gland, while the effects on many non-classical tissues (such as the kidney or heart) remain relatively unexplored.
The Zacharewski lab uses both in vivo and in vitro models to look at the effects of estrogen on gene expression in various tissues and cell types. Custom cDNA microarrays are used as wide-ranging genomic monitors to allow us to gain better insight into which genes are being regulated as a result of estrogenic action.
Projects for the term (January 2004 to June 2004):
Using cDNA microarrays, my main task during the term was to observe the changes of gene expression in murine heart and kidney as a result of estrogenic action. Mice were treated once or 3X24 by oral gavage with ethynyl estradiol (EE, an estrogenic compound), or vehicle. Organs of interest were harvested 2, 4, 8, 12, 18, 24, and 72 hours after the initial dose. Total RNA was isolated from the hearts and kidneys of the EE-treated (EET) and vehicle-treated (VT) mice from each time-point.Samples of EET and VT RNA from individual animals from each time-point were used to produce differentially labeled (Cy3-UTP or Cy5-UTP) cDNA probes via modified reverse transcription reactions. These probes were then pooled together and used for hybridization on our microarrays. Thus, each array represented a comparison of gene expression between EET and VT individuals at a specific time-point. In total, three sets of EET and VT individuals at each time-point were looked at.
Projects for the term (September 2004 to December 2004):
1) TCDD Mouse Uterus Time-Course Microarrays
Three replicates of cDNA microarray assays done to assess the gene expression changes in the murine uterus resulting from acute treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin. Because the mouse uterus yields such small amounts of total RNA, an indirect labeling approach was taken because of its higher sensitivity.
2) Verification of Estrogen Mouse Uterus Time-Course Microarray Results using quantitative real-time PCR (QRT-PCR).
QRT-PCR was performed on 16 active genes (as determined from previous microarray results), and results were compared to the microarray results to ensure correlation between the two.
3) PCB126 Rat hepatoma (H4IIe) cell Dose Response Microarrays
H4IIe cells were dosed with increasing concentrations of PCB126 and cDNA microarrays were used to gauge how increasing doses of the compound affect global gene expression.
Minor Projects and Tasks:
- Assisted in in vivo studies.
- Assisted in maintenance of cultured cells.
- RNA extractions from various mouse tissues.
- Use of QRT-PCR to detect a wide range of human nuclear receptors in stem cell lines.
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