Trp1-cre Revisited ; Evidence For Cre-recombinase Toxicity In The Retinal Pigment Epithelium

Abstract

Purpose: : The Cre/loxP recombinase system has been very useful in performing conditional gene targeting experiments to explore genetic pathways of the retinal pigment epithelium (RPE). However, recent studies have shown that Cre expression can be toxic to mammalian cells and we sought to explore this possibility in the Trp1-Cre mouse, which expresses Cre-recombinase under the tyrosinase related protein-1 promoter.

Methods: : For all the experiments described mice carrying the Cre transgene were identified by DNA tail biopsy and PCR analysis using the following primers. Littermates non-carrying the transgene were used as controls. All mice harboring the Trp1-Cre transgene were genotyped for the rd1 (retinal degeneration 1) mutation that was present in the founder mouse of FVB/N background, and mice that did not carry the rd1 mutation were used. Cre expression in the RPE was confirmed by crossing the Trp1-Cre mouse with the ROSA26R reporter mouse and subsequent X-Gal staining. To study the morphology of the RPE monolayer, we perfomed immunofluorescence studies using antibodies against β-catenin (Millipore) and ZO-1 (Invitrogen). Fluorescein angiography (FA) was performed using a commercial camera and imaging system (TRC 50 VT camera and IMAGEnet 1.53 system; Topcon, Paramus, NJ). Photographs were captured with a 20 D lens in contact with the fundus camera lens after intraperitoneal injection of 0.1 mL of 2% fluorescein sodium.

Results: : Macroscopic evaluation of flatmounts obtained from Trp1-Cre mice revealed large RPE defects mostly confined to the periphery of the eye. Fluorescein angiography revealed window defects most commonly located between 10 and 2 o’clock meridians as well as diffuse background hyperfluorescence. Immunofluorescent staining with the membrane markers ZO-1 and β-catenin revealed significant disorganization of the RPE layer with loss of the classic "honeycomb" appearance and enlarged, asymmetric RPE cells. The choroid could be visualized beneath the enlarged RPE cells stained with Concanavalin A lectin.

Conclusions: : Our data indicate that Cre expression in the Trp-1 Cre mouse results in significant toxicity in the RPE. This has implications regarding the interpretation of data obtained using this transgenic mouse

Keywords: retinal pigment epithelium