Excision of Individual Genes in RPE Cell Primary Cultures by Lentiviral–CRE

Abstract

Abstract: : Purpose: To establish a system for studying the roles of individual proteins in the RPE by using viral–CRE deletion of floxed genes. We have shown previously that myosin VIIa is required for normal delivery of phagosomes to the basal RPE, and hence, normal digestion. A specific longer–term goal of the present study is to test whether a microtubule motor, such as kinesin–1 or kinesin–2, works in concert with myosin VIIa in phagosome delivery. Since the general knockout of genes encoding many of the components of these motors is lethal, ad hoc CRE–lox recombination provides a valuable method for targeted gene deletion. Methods: Primary cultures of RPE cells were prepared from mice, including mice carrying floxed Kif3a and Kif5a genes. The cells were grown on Transwell polycarbonate filters, plastic, or glass. They were infected with lentiviral–CRE–GFP, and analyzed for CRE expression and Kif3a or Kif5a gene deletion. Results: CRE recombinase was detected in infected RPE cells by fluorescence of GFP as well as immunodetection of CRE. It was first evident after 24 hrs, reaching a maximum after 48 hrs. Removal of proteins encoded by floxed genes followed, depending on the half–life of the protein. Conclusions: Treatment of primary cultures of RPE cells with lentiviral–CRE provides a mechanism to test specific gene function, with gene excision under fairly precise temporal control.

Keywords: retinal pigment epithelium • transgenics/knock-outs • protein structure/function