Expression profiling by high throughput sequencing
Summary
In future high-throughput transcriptomic studies, we propose that the use of cell lines nullizygous for important targets of environmental chemicals will greatly facilitate the interpretation of regulated genes. To provide proof of this concept, we generated transcript profiles from wild-type and MTF-1-null cells exposed to zinc (Zn) and six putative activators of MTF-1 generated using TempO-Seq targeted sequencing of ~3000 human genes. In the absence of chemical exposure, knockout of MTF-1 resulted in expression changes in 44 genes indicating that MTF-1 has some effect on gene expression in the absence of overt activation. Almost all of the genes regulated by Zn and phenoxybenzamine were no longer regulated in the MTF-1-null cells. For the rest of the chemicals, more than half of the genes regulated in the wild-type cells were no longer regulated in the MTF-1-null cells. These studies demonstrate that many of the changes in gene expression after exposure to the putative MTF-1 activators were MTF-1-dependent.
Overall design
An initial concentration-range finding experiment was conducted in MCF-7 cells to determine the optimal concentration of the test chemicals to use in the study. Cells were plated in 12-well plates and incubated 48 hours. The media was replaced with dosing solutions containing DMSO (0.05%) or 1-100 uM of each chemical in DMSO (final concentration 0.05%). After 6 hours of exposure, media was removed and cells were lysed in 0.5 mL Trizol, followed by RNA extraction. Gene expression changes in wild-type and MTF1-null cells were evaluated using the human 1500+ Tempo-Seq platform (BioSpyder, Inc, Carlsbad, CA).
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