Figure 1. Generation, identification, and Rpe65 expression analysis of Rpe65^CreERT2 mice.

(A) KI strategy to introduce a P2A-CreER^T2 coding sequence in-frame with the final coding exon (exon 14) of the Rpe65 gene. Primer-binding sites and expected PCR product sizes are shown below the wild-type and targeted alleles. The neomycin cassette in the targeting vector was removed during expansion of the embryonic stem cell clone. FLP-recombinase was subsequently bred out by crossing with C57BL/6 mice. The KI allele allows cotranslational expression of RPE65-P2A and CreER^T2 as 2 separate polypeptides. The modified RPE65 protein contains an additional G⁵³⁴SGATNFSLLKQAGDVEENPG⁵⁵⁴ polypeptide sequence at its C-terminus, while CreER^T2 contains an additional Pro residue at its N-terminus. (B) Genotyping results from wild-type, Cre^+/–, and Cre^+/+ animals. (C) Western blot analysis of RPE65 expression. After normalization to the α-tubulin loading controls, the RPE65 expression levels in Cre^+/– and Cre^+/+ mice were estimated to be 59.7% and 1.1% of that for wild-type mice.