ASSEMBLY OF A GAL4-PBID EXPRESSION PLASMID FOR ANALYSIS OF PROMOTER ACTIVITY
Dylan Ochoa, Enrique Massa.
Texas A&M University-Kingsville, Kingsville, TX.
Analysis of gene regulatory elements is approached using various in vitro and in vivo methods. In vivo analysis of gene expression has routinely been performed utilizing Drosophila melanogaster as the model organism. In particular, targeting plasmids have been employed to target the fly genome for the analysis of gene regulatory elements using P-element-based expression plasmid constructs such as pPTGAL4. Recent developments have yielded new classes of targeting constructs that rely on distinct DNA elements that, in turn, allow for site-specific integration of the targeting constructs in the genome at a landing pad. Currently, there are no suitable constructs available for GAL4-driven expression of reporter genes in an insulated system that also allows for site-specific integration into the genome. In light of this, we have constructed a hybrid GAL4-based expression plasmid that provides an insulated background, allows for cloning of promoter fragments using a multiple cloning site, and allows for site-specific integration into the genome. This construct was generated by PCR amplification and cloning a GAL4-multiple cloning site DNA fragment into pBID, a site-specific integration plasmid. The functionality of this plasmid will be tested using the Drosophila KCNQ potassium channel promoters, which have been previously characterized and thus serve as a control set of promoters for characterization of this expression plasmid. The development of this plasmid will facilitate the analysis of gene promoter activity in an insulated background with a well-defined genomic location. This will provide a new tool for the analysis of gene activity in organisms such as Drosophila melanogaster.