An inducible Cre mouse for studying roles of the RPE in retinal physiology and disease.

Elliot H Choi,Minzhong Yu,Philip D Kiser,Zhiqian Dong,Steven J Fliesler,Seth Blackshaw,David E Einstein,Henri Leinonen,Krzysztof Palczewski,Sriganesh Ramachandra Rao,Neal S Peachey,Susie Suh

doi:10.1172/jci.insight.146604

Abstract

The retinal pigment epithelium (RPE) provides vital metabolic support for retinal photoreceptor cells and is an important player in numerous retinal diseases. Gene manipulation in mice using the Cre-LoxP system is an invaluable tool for studying the genetic basis of these retinal diseases. However, existing RPE-targeted Cre mouse lines have critical limitations that restrict their reliability for studies of disease pathogenesis and treatment, including mosaic Cre expression, inducer-independent activity, off-target Cre expression, and intrinsic toxicity. Here, we report the generation and characterization of a knockin mouse line in which a P2A-CreERT2 coding sequence is fused with the native RPE-specific 65 kDa protein (Rpe65) gene for cotranslational expression of CreERT2. Cre+/– mice were able to recombine a stringent Cre reporter allele with more than 99% efficiency and absolute RPE specificity upon tamoxifen induction at both postnatal days (PD) 21 and 50. Tamoxifen-independent Cre activity was negligible at PD64. Moreover, tamoxifen-treated Cre+/– mice displayed no signs of structural or functional retinal pathology up to 4 months of age. Despite weak RPE65 expression from the knockin allele, visual cycle function was normal in Cre+/– mice. These data indicate that Rpe65CreERT2 mice are well suited for studies of gene function and pathophysiology in the RPE.

Highlights

The retinal pigment epithelium (RPE) is a monolayer of cells that closely interacts with the photoreceptors of the retina and plays essential roles in the maintenance of visual function [1,2,3]
Our initial list included RPE-specific 65 kDa protein (Rpe65), lecithin:retinol acyltransferase (Lrat), Best1, and bestrophin 2 (Best2) [32]. Of these 4 genes, Rpe65 exhibited the highest mRNA expression and had the added advantage of being RPE specific, as opposed to the other 3 genes, which are expressed in nonretinal tissues, including the brain (Best1 and Best2) and liver (Lrat) [33]
We generated a KI mouse line with a P2A-CreERT2 sequence fused in-frame after the last coding exon of the native Rpe65 gene, which encodes the retinoid isomerase of the classical visual cycle [34] (Figure 1A)

Summary

Introduction

The retinal pigment epithelium (RPE) is a monolayer of cells that closely interacts with the photoreceptors of the retina and plays essential roles in the maintenance of visual function [1,2,3]. The RPE critically supports the photochemistry of vision by supplying the visual chromophore, 11-cis-retinal, to rod and cone opsins, thereby generating functional visual pigments [4, 5]. It protects photoreceptors by forming the outer blood-retinal barrier, allowing selective transport of nutrients and waste products between the photoreceptor cells and the systemic circulation, and contributes to the maintenance of the photoreceptor outer segments [6] and the choroid [7]. Genetic factors are pivotal in the disease etiology and pathogenesis [18]

Methods

Results

Conclusion