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

ABSTRACT: To dissect gene functions in the retinal pigment epithelium (RPE), we previously generated a tetracycline-inducible RPE-specific Cre mouse line. Although this Cre mouse line was useful for several conditional gene targeting studies that were conducted by different laboratories, its potential has not been fully exploited, presumably due to a lack of knowledge or procedure for inducing Cre expression appropriately in this mouse line. The goal of the current study is to establish a procedure that will improve the reproducibility of Cre-mediated recombination in this mouse line.Analysis of Cre expression and function was performed in double transgenic mice derived from inducible RPE-specific Cre and Cre-activatable ROSA26 lacZ reporter mice. A tetracycline derivative, doxycycline, was supplied to mice intravitreally to induce Cre expression. Cre expression and function were examined with reverse transcription-PCR, immunoblotting, immunostaining, and in situ enzymatic assay for ?-galactosidase. Retinal integrity was examined with electroretinography and morphometry.Intravitreal Dox injection elevated Cre expression significantly and resulted in productive Cre-mediated recombination in approximately 60% of the RPE cells in this mouse line with no apparent change in retinal integrity.Our results suggest that productive Cre-mediated recombination in this mouse line can be induced efficiently with intravitreal Dox delivery, with no apparent Dox or 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 Cre mouse line in long-term studies requiring conditional gene targeting.

SUBMITTER: Fu S 

PROVIDER: S-EPMC3984040 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature