NOD/ShiLtJ-Gt(ROSA)26Sor^{em1.1(CAG-cas9*,-EGFP)Dvs}/J

Stock number: 030936
Product name: NOD Rosa Cas9 Constitutive
Strain synonyms: NOD Rosa26^{CAG-Cas9-P2A-EGFP} knock-in
Research areas: Neurobiology Research, Research Tools

Description

NOD Rosa26^{CAG-Cas9-P2A-EGFP} knock-in mice constitutively express CRISPR associated protein 9 endonuclease (cas9) 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

The Gt(ROSA)26Sor^{em1.1(CAG-cas9*,-EGFP)Dvs} knock-in strain (Rosa26^{CAG-Cas9-P2A-EGFP}) is expected to have widespread expression of cas9 and EGFP.

Whereas gene editing via viral delivery of cas9 is burdened by packaging size limits, these Rosa26^{CAG-LSL-Cas9-P2A-EGFP} mice only require the user to select a specific guide sequence for generating single or multiple simultaneous mutations.

Mice homozygous for the Rosa26^{CAG-Cas9-P2A-EGFP} knock-in allele are viable and fertile. Mice on the NOD/ShiLtJ inbred background (Stock No. 001976) develop spontaneous autoimmune Type 1 diabetes. As such, these Rosa26^{CAG-Cas9-P2A-EGFP} mice (both homozygous and heterozygous) should be given CFA prior to six weeks of age to delay diabetes onset.

Of note, Stock No. 030936 is currently being assessed for diabetic phenotype (January 2019). As the mice are characterized, we will modify the strain description and add phenotype data.

Development

The Gt(ROSA)26Sor^{em1.1(CAG-cas9*,-EGFP)Dvs} knock-in allele was created by the Type 1 Diabetes Repository at The Jackson Laboratory by microinjecting Cre recombinase mRNA into NOD/ShiLtJ-Gt(ROSA)26Sor^{em1(CAG-cas9*,-EGFP)Dvs}/DvsJ mouse zygotes (Stock No. 027891) to remove the floxed STOP cassette.

To generate the Gt(ROSA)26Sor^{em1(CAG-cas9*,-EGFP)Dvs} allele, CAG-LSL-Cas9-P2A-EGFP-WPRE-BGHpA sequences were obtained from Feng Zhang (Massachusetts Institute of Technology). Specifically, these sequences include CMV enhancer/chicken beta-actin promoter (CAG), a loxP-STOP(3xSV40 polyA)-loxP cassette (LSL), a 3xFLAG sequence, a mammalian codon-optimized cas9 gene (derived from Streptococcus pyogenes CRISPR associated protein 9 [SpCas9]), a P2A ribosomal skip cleavage peptide sequence, an enhanced green fluorescent protein gene (EGFP), a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and a bovine growth hormone polyA sequence (BGHpA). To facilitate insertion into the Gt(ROSA)26Sor locus on chromosome 6 via homology directed repair (HDR), the LSL-Cas9-P2A-EGFP sequences were co-injected with a large piece of Gt(ROSA)26Sor locus homologous DNA. Founder mice were bred to NOD/ShiLtJ inbred mice (Stock No. 001976), and then heterozygous mice were bred to NOD/ShiLtJ for one additional generation while characterizing the mutation. Rosa26^{CAG-LSL-Cas9-P2A-EGFP} heterozygous mice were then bred together to produce homozygous mice.

NOD/ShiLtJ-Gt(ROSA)26Sor^{em1(CAG-cas9*,-EGFP)Dvs}/DvsJ (Stock No. 027891) homozygous zygotes were microinjected with mRNA for Cre recombinase. Correctly targeted embryos were transferred to pseudopregnant females. Correctly targeted pups were identified by sequencing and PCR and further bred to NOD/ShiLtJ inbred mice (Stock No. 001976) to develop the colony. Heterozygotes were intercrossed to generate homozygotes.

Allele

Symbol: Gt(ROSA)26Sor^{em1.1(CAG-cas9*,-EGFP)Dvs}
Name: endonuclease-mediated mutation 1.1, David V Serreze
MGI accession ID: MGI:6272395
Generation method: Endonuclease-mediated
Attributes: Reporter; Endonuclease
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: NOD/ShiLtJ
Site of expression: EGFP and Cas9 are expected to have widespread expression.
Molecular note: Cre-mediated recombination removed the upstream floxed STOP cassette from Gt(ROSA)26Sor^{em1(CAG-cas9*,-EGFP)Dvs}.