B6J.129(Cg)-Gt(ROSA)26Sor^{tm1.1(CAG-cas9*,-EGFP)Fezh}/J

Stock number: 026179
Product name: Rosa26-Cas9 knockin on B6J
Research areas: Neurobiology Research, Research Tools

Description

Rosa26-Cas9 knock-in mice constitutively express CRISPR associated protein 9 (cas9) endonuclease and EGFP in a widespread fashion under the direction of a CAG promoter. When used in combination with single guide RNAs, they allow editing of single or multiple mouse genes in vivo or ex vivo.

Detailed description

Rosa26-Cas9 knock-in mice have a CAG promoter directing the widespread expression cas9 and EGFP. EGFP fluorescence is widespread in brain, kidney, liver, spleen and heart tissue, and Cas9 protein is detected in whole brain. Analysis of multiple organs (including brain, kidney, liver, spleen, heart tissue) show that constitutive cas9 expression does not result in any detectable toxicity, morphological abnormalities, tumor formation, or up-regulation in DNA damage or apoptosis markers. Whereas gene editing via viral delivery of cas9 is burdened by packaging size limits, these Rosa26-Cas9 mice only require a specific single guide RNA (sgRNA) for generating single or multiple simultaneous mutations. Mice homozygous for the Rosa26-Cas9 knock-in allele on the STOCK and congenic C57BL/6J backgrounds are viable and fertile. Some mice have a white belly spot.

In an attempt to offer alleles on well-characterized or multiple genetic backgrounds, alleles are frequently moved to a genetic background different from that on which an allele was first characterized. This is the case for the strain above. It should be noted that the phenotype could vary from that originally described. We will modify the strain description if necessary as published results become available.

Rosa26-Cas9 knock-in mice are also available on a C57BL6/NJ background (Stock No. 026430), on a FVB/NJ congenic background (Stock No. 026558), on a congenic NOD/ShiLtJ background (Stock No. 026557), as well as a STOCK background (Stock No. 024858).

Development

To create the Rosa26-Cas9 knock-in allele, the Rosa26-LSL-Cas9 targeting vector was used. This vector was designed with (from 5' to 3') a 5' homology arm, a ubiquitously expressed CAG promoter, loxP-flanked 3xSV40 polyA stop cassette, a 3X-FLAG epitope tag, a mammalian codon-optimized cas9 gene (derived from Streptococcus pyogenes CRISPR associated protein 9 [SpCas9]) flanked by two nuclear localization signals, a P2A ribosomal skip cleavage peptide sequence, an enhanced Green Fluorescent Protein (EGFP) gene, a woodchuck hepatitis virus post-transcriptional regulatory element, bovine growth hormone polyA, pPGK-Neo-pA positive selection cassette and a 3' homology arm. The construct was electroporated into 129-derived R1 embryonic stem (ES) cells. Correctly targeted ES cells were injected into C57BL/6N blastocysts. The resulting chimeric animals were tested for germline transmission by crossing to C57BL/6N mice. The mice were then crossed to a beta-actin driven Cre recombinase strain on the FVB background (Stock No. 003376) to excise the floxed STOP cassette. Upon arrival at The Jackson Laboratory, the mice were crossed to C57BL/6J (Stock No. 000664) at least once to establish the colony. The mice were then backcrossed to C57BL/6J (Stock No. 000664) using a marker assisted protocol.

In 2018, 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 rederived living colony at The Jackson Laboratory Repository. All markers tested were C57BL/6J allele-type with a single exception - one marker on Chromosome 11 [~21 Mb] is segregating with 129 allele-type.

Again, in 2019, a 48 SNP was run on this colony of mice. While the one marker on Chromosome 11 was shown to be C57BL/6J-like, a marker on Chromosome 6 was segregating with either 129, B6N, or FVB, suggesting an incomplete backcross.

Allele

Symbol: Gt(ROSA)26Sor^{tm1.1(CAG-cas9*,-EGFP)Fezh}
Name: targeted mutation 1.1, Feng Zhang
MGI accession ID: MGI:5583838
Generation method: Targeted
Attributes: Reporter; Endonuclease
Synonyms: constitutive Cas9; Gt(ROSA)26Sor^{tm1.1(CAG-cas9,-EGFP)Fezh}; Rosa26-Cas9
Marker symbol: Gt(ROSA)26Sor
Marker name: gene trap ROSA 26, Philippe Soriano
Marker synonyms: beta geo; Gtrgeo26; Gtrosa26; R26; ROSA26; SETD5-AS1; Thumpd3as1
Chromosome: 6
Strain of origin: (129X1/SvJ x 129S1/Sv)F1-Kitl⁺
Expressed genes: cas9,CRISPR associated protein 9,; GFP,Green Fluorescent Protein,
Site of expression: EGFP and cas9 will be expressed in a widespread fashion under the direction of a CAG promoter
Molecular note: The targeting vector inserted into the locus was designed with (from 5' to 3') a 5' homology arm, a ubiquitously expressed CAG promoter, loxP-flanked 3xSV40 polyA stop cassette, a 3X-FLAG epitope tag, a mammalian codon-optimized cas9 gene (derived from Streptococcus pyogenes CRISPR associated protein 9 [SpCas9]) flanked by two nuclear localization signals, a P2A ribosomal skip cleavage peptide sequence, an enhanced Green Fluorescent Protein (EGFP) gene, a woodchuck hepatitis virus post-transcriptional regulatory element, bovine growth hormone polyA, pPGK-Neo-pA positive selection cassette and a 3' homology arm. Cre-mediated recombination removed the floxed STOP cassette.