C57BL/6-Rpe65^{tm1.1(cre/ERT2)Kser}/J

Stock number: 035973
Product name: Rpe65^CreERT2
Research areas: Developmental Biology Research, Research Tools, Sensorineural Research

Description

Rpe65^CreERT2 knock-in mice have a P2A-Cre^ERT2 coding region fused in-frame after the last amino acid-encoding codon of the endogenous retinal pigment epithelium 65 (Rpe65) gene. They are useful for studying gene function and pathophysiology of retinal diseases in the retinal pigment epithelium.

Detailed description

Rpe65^CreERT2 knock-in mice have a P2A-Cre^ERT2 coding region fused in-frame after the last amino acid-encoding codon of the endogenous retinal pigment epithelium 65 (Rpe65) gene. Rpe65 encodes a protein expressed specifically in the retinal pigment epithelium (RPE), a monolayer of cells that closely interacts with the photoreceptors of the retina and plays essential roles in the maintenance of visual function. Homozygous mice are viable and fertile.

When these mice are bred with mice containing loxP-flanked sequences, tamoxifen-inducible Cre-mediated recombination will result in deletion of the floxed sequences in the RPE. Heterozygous Rpe65^CreERT2 mice recombine with the mT/mG reporter (Stock No. 007676) with >99% efficiency and absolute RPE specificity upon tamoxifen induction at both post-natal day 21 and day 50. Induced heterozygous Rpe65^CreERT2 mice exhibit no signs of structural or functional retinal pathology up to 4 months of age. Tamoxifen-independent Cre activity is negligible. RPE65 expression in heterozygous Rpe65^CreERT2 mice is ~60% that of wild-type mice whereas expression in homozygous Rpe65^CreERT2 mice is much weaker (~1%). Homozygous Rpe65^CreERT2 mice exhibit reductions in dark-adapted electroretinogram (ERG) responses that are attributable to slowed visual cycle kinetics, whereas heterozygous Cre^+/- mice had normal ERG and visual chromophore recovery profiles. Heterozygous Rpe65^CreERT2 mice are recommended for conditional knockout studies.

Development

The Rpe65^CreERT2 targeting construct was designed to fuse a P2A self-cleaving peptide sequence and a cre/ERT2 sequence in frame at the 3’ end of exon 14 of the retinal pigment epithelium 65 (Rpe65) gene. The cre-ERT2 fusion gene contains a Cre recombinase fused to a human estrogen receptor ligand binding domain. A frt-flanked neomycin resistance (neo) cassette was placed immediately after the non-coding exon 15. This construct was electroporated into C57BL/6 FLP-derived iTL BF1 embryonic stem (ES) cells. The Neo cassette in the targeting sequence was removed during ES clone expansion. Correctly targeted ES cells were injected into BALB/c blastocysts and the chimeric offspring were bred to C57BL/6N mice to generate germline neo cassette-deleted mice. The resulting Rpe65^CreERT2 mice were bred to C57BL/6N mice to remove the Flp-expressing transgene. The donating lab subsequently bred these mice to C57BL/6J mice for 2 generations to remove the C57BL/6N-derived Rd8 gene mutation found in all C57BL/6N substrains. Upon arrival at The Jackson Laboratory, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).

In 2021, a 48 SNP (single nucleotide polymorphism) panel analysis, with 43 markers covering all 19 chromosomes and the X chromosome, as well as 5 markers that distinguish between the C57BL/6J and C57BL/6N substrains, was performed on the males sent to The Jackson Laboratory Repository. Two of the 5 markers that determine C57BL/6J from C57BL/6N were found to be segregating on Chromosomes 8 and 19. The rd8 recessive mutation associated with retinal degeneration, on Chromosome 1 within the Crb1 locus, was determined to be C57BL/6J like. These data suggest the mice sent to The Jackson Laboratory Repository were on a mixed C57BL/6N;C57BL/6J genetic background.

Allele

Symbol: Rpe65^{tm1.1(cre/ERT2)Kser}
Name: targeted mutation 1.1, Philip Kiser
MGI accession ID: MGI:6501720
Generation method: Targeted
Attributes: Recombinase-expressing; Inducible
Marker symbol: Rpe65
Marker name: retinal pigment epithelium 65
Marker synonyms: A930029L06Rik; BCO3; LCA2; Mord1; mRPE65; p63; rd12; RP20; sRPE65
Chromosome: 3
Strain of origin: C57BL/6
Molecular note: The targeting construct was designed to fuse a P2A self-cleaving peptide sequence and a cre/ERT2 sequence in frame at the 3' of exon 14 of the retinal pigment epithelium 65 (Rpe65) gene. The cre-ERT2 fusion gene contains a Cre recombinase fused to a human estrogen receptor ligand binding domain. A frt-flanked neomycin resistance (neo) cassette was placed immediately after the non-coding exon 15. This construct was electroporated into C57BL/6 FLP-derived iTL BF1 embryonic stem (ES) cells. The Neo cassette in the targeting sequence was removed during ES clone expansion.