Evaluating Molecular Function of Proteins Through Integrase-Mediated Cassette Exchange

Friday, 13 February 2015
Exhibit Hall (San Jose Convention Center)
Sanjay V. Kottapalli, Clarendon Hills, IL
One method for investigating protein function involves lowering expression of the endogenous protein by RNA interference (knockdown) in order to examine the resulting cellular phenotype. However, problems arise when attempting to “rescue” the phenotype by reintroducing mutants of the protein at its original expression level, due to position effects. Here, we introduce a method for site-specific integrase-mediated cassette exchange (IMCE), which utilizes the enzyme ΦC31 Integrase to swap the genetic material between attP sites on an acceptor cassette with the DNA between attB sites on a donor vector. Our acceptor cassette was constructed with the phosphoglycerate kinase promoter and the mCherry red fluorescent protein gene, flanked by attP sites. The donor cassette was constructed with the Enhanced Green Fluorescent Protein (EGFP) gene, flanked by attB sites. A cotransfection of the acceptor and donor cassettes +/- Integrase into cells showed several instances of EGFP expression, indicating moderate success of IMCE between transient plasmids. These preliminary results show promise for planned knockdown/rescue experiments involving cotransfections of Integrase and donor cassettes incorporating rescue transgenes in cells containing the acceptor cassette integrated at a stable locus. This method will have broader uses in molecular analyses by ensuring uniform expression of wildtype and mutant proteins.