Analysis of the J alpha-C alpha Introns Function in Transgenic Mice
A locus control region (LCR) is a cis-acting gene regulatory element. It can be used to direct tissue-specific, copy number-dependent expression of a linked gene(s) that is independent of the genomic site of integration. The mouse TCRα LCR located within the endogenous TCRα/δ/Dad1 locus is a prime example of an LCR. It produces largely T cell restricted gene expression at levels that are transgene copy number-dependent. The endogenous TCRa gene is highly expressed in both thymic and peripheral T cells. However, previous studies examining TCRα LCR activity in bacterial artificial chromosome (BAC) reporter transgenic mice have yet to reveal the mechanisms driving high-level TCRα expression in peripheral T cells. Preliminary data implicate the Jα-Cα intron sequence as playing a role in TCRa expression in peripheral T cells. However, this region has yet to be directly explored for gene regulatory activity. We will be studying gene expression from a wild type TCRa/LCR/Dad1 reporter BAC construct (WT BAC), as well as a deletion mutant version of the BAC in which the Jα-Cα sequence is removed from the WT BAC (mutant BAC). We will first be determining baseline expression levels of reporter gene (representing TCRa) in the wild type BAC. We will collect this data from thymus and peripheral (spleen) T cells as well as in B cells. While doing so, we will take note of the stage at which reporter expression begins. For the WT BAC, we expect to see high-level reporter gene expression in both the thymic and spleen T cells; as well as little to no reporter expression in B cells. Once we have this baseline we will then compare relative reporter gene expression levels of wild type and mutant BAC in the various lymphocyte populations. These experiments will reveal the function of the Ja-Ca intronic DNA sequence in TCRa gene regulation.
Supported by NIH-RCMI grant numbers MD007599 (formerly RR003037) and NIDA grant R25DA032520.