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
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]
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