Jennifer Cunningham
Department of Biology
Marquette University
Dr. Stephen Munroe

The gene erbA-alpha is alternatively spliced to form two different mRNA molecules. These mRNAs code for two major thyroid hormone receptor (TR) isoforms called TRa1 and TRa2. These receptor isoforms are functionally antagonistic. The regulation of this alternative splicing is important in determining cellular levels of TRa1 and TRa2.  In relation to these isoforms, it was found that another gene, RevErbA-alpha (RevErb) is also transcribed from the opposite direction at the same locus and it overlaps the TRa2 coding region. Further studies showed coexpression of RevErb and TRa2  regulates the TRa1/TRa2 ratio in cells.

My research is focusing on establishing a cell line which expresses RevErb, TRa1, and TRa2 in a regulated manner.  The C6 cell line will be regulated by the presence or absence of tetracycline.  The Tet-Off System will be used to express the gene of interest, which is RevErb. The transcription of this gene is induced in the absence of tetracycline. In the presence of tetracycline, transcription is turned off. The first critical step is to get the pTet-Off regulatory plasmid stably transfected into the C6 cells. The stable cell lines will be screened by performing transient transfection assays with the reporter vector pBI-EGFP-Luc.Use of the GFP reporter is important because it will be used to test for tet-off regulation as well as expression of RevErb. GFP expression is also easily detected by fluorescence microscopy. After being screened, the Tet-Off cell line will then be used for transfection of the gene of interest. 

C6 cells are rat glioma cells which have an endogenous expression of a greater amount of TRa2 than TRa1. My goal is to transfect the Tet-Off C6 cells with a plasmid construct which will include the RevErb gene, and then to screen for RevErb expression in the presence and absence of tetracycline by using a gene specific luciferase assay system.  RevErb expression in the absence of tetracycline can then be compared with its endogenous expression.