Characterization of Cre Transgenic Mice Achieving Rpe-Specific Expression/Knockout of Floxed Sequences, Using the Cre-Loxp System

Abstract

Purpose: : The Cre-loxP system can be used to create conditional knockout mice. Our goal is to create an RPE-specific Cre expressing transgenic mouse that can be used to generate a cell-type specific knockout. This will facilitate testing of cell-autonomous gene function and enable study of genes whose systemic absence is lethal. A new transgenic mouse line with VMD2 promoter driven Cre expression allows the conditional removal of genes in the RPE.

Methods: : Transgenic mice expressing Cre-recombinase in the RPE were generated by placing the Cre recombinase gene under control of the Vmd2 promoter. Cre expression was assessed using Immunofluorescence (IF) in 3, 9 and 18 month old mice. Additionally, Cre mediated excision of a floxed sequence was analyzed using PCR analysis of RPE/Choroid and Neural Retina tissue.

Results: : VMD2-Cre mice demonstrated Cre expression in RPE cells by IF staining. The number of Cre positive RPE cells varied among mice derived from a single founder but was always greater than 50% of total RPE cells. PCR analysis of RPE/Choroid and Neural Retina tissues demonstrated excision of floxed sequence in Cre+ RPE/Choroid tissue, and absence of excision in Cre+ Neural Retina, as well as all Cre- tissues.

Conclusions: : The VMD2-Cre line demonstrates Cre expression in greater than 50% of RPE cells by IF. Additionally, this line achieves RPE specific excision of floxed sequence through the utilization of VMD2 promoter, as demonstrated by PCR analysis. Morphologic analysis suggests no Cre toxicity. This line should be useful for studies or RPE autonomous gene function, and Cre- cells provide an internal control.

Keywords: retinal pigment epithelium