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.
Feng Zhang, Massachusetts Institute of Technology
Genetic Background | Generation |
---|---|
000664 C57BL/6J |
N5F6
|
Allele Type | Gene Symbol | Gene Name |
---|---|---|
Targeted (Reporter, Endonuclease) | Gt(ROSA)26Sor | gene trap ROSA 26, Philippe Soriano |
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).
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.
Expressed Gene | GFP, Green Fluorescent Protein, |
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Expressed Gene | cas9, CRISPR associated protein 9, |
Site of Expression | EGFP and cas9 will be expressed in a widespread fashion under the direction of a CAG promoter |
Allele Name | targeted mutation 1.1, Feng Zhang |
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Allele Type | Targeted (Reporter, Endonuclease) |
Allele Synonym(s) | constitutive Cas9; Gt(ROSA)26Sortm1.1(CAG-cas9,-EGFP)Fezh; Rosa26-Cas9 |
Gene Symbol and Name | Gt(ROSA)26Sor, gene trap ROSA 26, Philippe Soriano |
Gene Synonym(s) | |
Promoter | CAG, CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter, |
Expressed Gene | GFP, Green Fluorescent Protein, |
Expressed Gene | cas9, CRISPR associated protein 9, |
Site of Expression | EGFP and cas9 will be expressed in a widespread fashion under the direction of a CAG promoter |
Strain of Origin | (129X1/SvJ x 129S1/Sv)F1-Kitl+ |
Chromosome | 6 |
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. |
When maintaining a live colony, these mice can be bred as homozygotes. Some mice may have a white belly spot.
When using the Rosa26-Cas9 knockin on B6J mouse strain in a publication, please cite the originating article(s) and include JAX stock #026179 in your Materials and Methods section.
Service/Product | Description | Price |
---|---|---|
Heterozygous for Gt(ROSA)26Sor<tm1.1(CAG-cas9*,-EGFP)Fezh> |
Frozen Mouse Embryo | B6J.129(Cg)-Gt(ROSA)26Sor<tm1.1(CAG-cas9*-EGFP)Fezh>/J | $2595.00 |
Frozen Mouse Embryo | B6J.129(Cg)-Gt(ROSA)26Sor<tm1.1(CAG-cas9*-EGFP)Fezh>/J | $2595.00 |
Frozen Mouse Embryo | B6J.129(Cg)-Gt(ROSA)26Sor<tm1.1(CAG-cas9*-EGFP)Fezh>/J | $3373.50 |
Frozen Mouse Embryo | B6J.129(Cg)-Gt(ROSA)26Sor<tm1.1(CAG-cas9*-EGFP)Fezh>/J | $3373.50 |
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The Jackson Laboratory has rigorous genetic quality control and mutant gene genotyping programs to ensure the genetic background of JAX® Mice strains as well as the genotypes of strains with identified molecular mutations. JAX® Mice strains are only made available to researchers after meeting our standards. However, the phenotype of each strain may not be fully characterized and/or captured in the strain data sheets. Therefore, we cannot guarantee a strain's phenotype will meet all expectations. To ensure that JAX® Mice will meet the needs of individual research projects or when requesting a strain that is new to your research, we suggest ordering and performing tests on a small number of mice to determine suitability for your particular project. We do not guarantee breeding performance and therefore suggest that investigators order more than one breeding pair to avoid delays in their research.
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