Inducible Gene Knockout System in the Retinal Pigmented Epithelium

Abstract

Abstract: : Purpose: Disruption of essential genes in mice often leads to embryonic and neonatal lethality. To circumvent this problem and dissect gene functions in the retinal pigmented epithelium (RPE), we decided to establish a temporal and spatial knockout system in the RPE using the Cre/lox system and the tetracycline–inducible gene expression technology. Methods: Transgenic constructs carrying a human bestrophin (VMD2) promoter controlled tetracycline responsive–transactivator (rtTA) gene or a tetracycline operator controlled Cre gene were co–injected to generate transgenic mice. RT–PCR analysis of rtTA was performed in mouse strains that had both rtTA and Cre integrated into a single chromosome to identify potentially useful strains. Inducible Cre expression was characterized with a functional assay using a Cre–activatable lacZ reporter mouse strain (R26R). Results: Most transgenic mice generated in this study had both rtTA and Cre transgenes integrated into a single chromosome. RT–PCR analysis of rtTA expression suggested that several transgenic strains expressed rtTA mRNA in the retina. ß–Galactosidase assay on whole mounts and sections of F1 mice derived from the rtTA/Cre mice and the R26R reporter mice suggested that at least one of these mouse strains was capable of carrying out productive Cre–mediated recombination in the RPE after induction of gene expression with doxycycline. Conclusions: We have generated transgenic mice that efficiently express Cre recombinase in the RPE in an inducible fashion. These mice can potentially be useful in gene function studies in the RPE.

Keywords: gene/expression • genetics • retinal pigment epithelium