Efficient induction of productive Cre-mediated recombination in the retinal pigment epithelium

Abstract

 

Purpose

 

To dissect gene functions in adult retinal pigmented epithelium (RPE) cells, we previously generated a tetracycline-inducible RPE-specific Cre mouse line (IOVS 49:1248, 2008). To improve the reproducibility of productive Cre-mediated recombination for inducible gene activation and inactivation in the RPE, we recently investigated the inducibility of Cre recombinase in this mouse line.

 

Methods

 

Analysis of Cre expression was carried out in double transgenic mice derived from inducible RPE-specific Cre and Cre-activatable lacZ reporter R26R mice. Tetracycline derivative, doxycycline (Dox), was supplied to mice intravitreally or intraperitoanlly. Cre expression/function was measured by enzymatic activity and immunohistochemcial staining for beta-galactosidase.

 

Results

 

While intraperitoneal Dox injection induced Cre-mediated recombination in some mice, intravitreal Dox delivery resulted in productive Cre-mediated recombination in at least 60% of RPE cells. In addition, a brief Dox induction did not cause any apparent alteration in retinal integrity.

 

Conclusions

 

Our results suggest that productive Cre-mediated recombination can be achieved efficiently in mice induced by intravitreal Dox delivery, with no apparent Cre toxicity. Therefore, our inducible RPE-specific Cre mice are suitable for Cre/lox-based gene activation and inactivation in adult RPE, which is critical to the effectiveness and suitability of this mouse line in long-term studies requiring conditional gene targeting.

 

 

Immunohistochemical analysis of Cre-activated reporter beta-galactosidase expression (green) in the RPE flat-mounts. Productive Cre-mediated recombination occurred in approximately 60% of the RPE cells.

 

Keywords: 701 retinal pigment epithelium • 533 gene/expression • 740 transgenics/knock-outs