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An inducible Cre mouse for studying roles of the RPE in retinal physiology and disease
Elliot H. Choi, … , Krzysztof Palczewski, Philip D. Kiser
Elliot H. Choi, … , Krzysztof Palczewski, Philip D. Kiser
Published March 30, 2021
Citation Information: JCI Insight. 2021;6(9):e146604. https://doi.org/10.1172/jci.insight.146604.
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Resource and Technical Advance Genetics Ophthalmology

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

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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.

Authors

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

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Figure 2

Cre recombinase activity in Rpe65CreERT2 mouse retina assessed with the mT/mG reporter.

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Cre recombinase activity in Rpe65CreERT2 mouse retina assessed with the ...
(A and B) RPE flatmounts from representative Cre+/– female and male mice, respectively, that were administered IP tamoxifen for 5 consecutive days starting on PD21 (n = 4). The flatmounts were obtained 2 weeks after completion of the tamoxifen regimen and were imaged with a fluorescence microscope. Green fluorescence indicates cells where Cre-mediated recombination has occurred, whereas red fluorescence indicates un-recombined cells. (C and D) RPE flatmounts from representative Cre+/– female and male mice that were administered IP tamoxifen for 5 consecutive days starting on PD50 (n = 7). The flatmounts were obtained 2 weeks after completion of the induction regimen. (E and F) show representative RPE flatmounts from Cre+/– female and male mice, respectively, that were not treated with tamoxifen (n = 6). The flatmounts were obtained on PD64. Scale bars indicate 500 μm. The insets in A–F show zoomed areas (marked by white squares, original magnification, 10×) with green and red channels separately displayed to demonstrate the single-cell resolution of the imaging method. (G and H) Retina cryosections from Cre+/– female and male mice, respectively, that were administered IP tamoxifen for 5 consecutive days starting on PD21 (n = 2). The cryosections were obtained 2 weeks after completion of the induction regimen. Insets show zoomed views of the areas marked with white rectangles with the retinal cell layers labeled. The green label represents Cre-mediated recombination and is restricted to the RPE. The scale bars in G and H indicate 500 μm while those in the insets indicate 100 μm. GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; OS, outer segment; RPE, retinal pigment epithelium; Tam, tamoxifen.

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