An inducible Cre mouse for studying roles of the RPE in retinal physiology and disease
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
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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Resource and Technical Advance Genetics Ophthalmology

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

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 4

In vivo visual chromophore recovery in Rpe65CreERT2 mice following a photobleach.

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In vivo visual chromophore recovery in Rpe65CreERT2 mice following a pho...
(A) Representative HPLC chromatograms showing separation of retinoids extracted from the eyes of a PD30 wild-type mouse (M450 wild-type Rpe65) and a Cre+/– littermate. The extracts were obtained after an in vivo 5000 lux × 10 minutes light exposure that bleached approximately 95% of rhodopsin followed by 4 hours of dark adaptation. Peaks “a” and “b” were identified as all-trans-retinyl esters and 11-cis-retinal oxime based on their retention times and absorbance spectra (inset). A solvent change artifact at approximately 21 minutes was omitted from all the chromatograms for clarity. (B) Quantification of all-trans-retinyl esters (left) and 11-cis-retinal oxime (right) in the extracts did not reveal a significant difference between the 2 groups (304.3 ± 23.6 vs. 352 ± 10.9, P = 0.09; and 158.5 ± 21.5 vs. 124.6 ± 12.2, P = 0.19, respectively). Statistical significance was assessed with 2-tailed t tests. (C) Representative HPLC traces showing separation of retinoids extracted from the eyes of a PD30 wild-type mouse (L450 wild-type Rpe65) and a Cre+/– littermate as well as an age-matched Cre+/+ mouse with the same genetic background. (D) Quantification of all-trans-retinyl esters (left) and 11-cis-retinal oxime (right) in the extracts did not reveal a significant difference between wild-type (L450, n = 9) and Cre+/– (n = 6) mice (147.2 ± 8.8 vs. 197.9 ± 31.1, P = 0.29; and 278.5 ± 13.3 vs. 268.3 ± 25.2, P = 0.89, respectively). All-trans-retinyl esters (562.6 ± 33.8) and 11-cis-retinal oxime (28.5 ± 3.6) levels in Cre+/+ mice were significantly different from both wild-type and Cre+/– mice (***P < 0.0001). Each point represents data from a single mouse. Bars indicate means ± SEM. Statistical significance was assessed by 1-way ANOVA followed by Tukey’s multiple comparisons test. ns, not statistically significant (P > 0.05).