A C57BL/6J congenic version of this strain is available as Stock No. 026175.
These CRISPR/Cas9 knockin mice have Cre recombinase-dependent expression of CRISPR associated protein 9 (cas9) endonuclease, a 3X-FLAG epitope tag and EGFP directed by a CAG promoter. Expression of Cas9 and EGFP is prevented by an upstream Lox-Stop-Lox (LSL) sequence. When used in combination with single guide RNAs and a Cre source, they allow editing of single or multiple mouse genes in vivo or ex vivo.
Feng Zhang, Massachusetts Institute of Technology
Genetic Background | Generation |
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|
Allele Type | Gene Symbol | Gene Name |
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Targeted (Conditional ready (e.g. floxed), Reporter, Endonuclease) | Gt(ROSA)26Sor | gene trap ROSA 26, Philippe Soriano |
These Rosa26-LSL-Cas9 knock-in mice have a floxed-STOP cassette preventing expression of the downstream bicistronic sequences (Cas9 and EGFP). Although under control of a CAG promoter, widespread expression of cas9 and EGFP is prevented by the STOP cassette. After exposure to Cre recombinase, expression of cas9 and EGFP is observed. The donating investigator reports cas9 expression is tightly controlled in a Cre-dependent manner. Whereas gene editing via viral delivery of cas9 is burdened by packaging size limits, these Rosa26-LSL-Cas9 mice only require one to select a Cre recombinase driven by the promoter of their choosing and a specific single guide RNA (sgRNA) for generating single or multiple simultaneous mutations. Mice homozygous for the Rosa26-LSL-Cas9 knock-in allele are viable and fertile.
The Rosa26-LSL-Cas9 knock-in mice are also available on a FVB/NJ congenic background (Stock No. 026481), a C57BL/6J congenic background (Stock No. 026175), a NOD/ShiLtJ congenic background (Stock No. 026431), as well as a C57BL/6NJ congenic background (Stock No. 026556).
The Rosa26-LSL-Cas9 targeting vector used 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 129S-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 backcrossed for four generations onto C57BL/6N. Upon arrival at The Jackson Laboratory, the mice were crossed to C57BL/6J (Stock No. 000664) at least once to establish the colony.
SNP (single nucleotide polymorphism) analysis performed as part of The Jackson Laboratory quality control effort indicates an incomplete backcross.
A 32 SNP panel analysis (27 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 live colony at The Jackson Laboratory Repository. Five of the 27 markers were segregating for 129. This suggests an incomplete backcross. All 5 markers that distinguish C57BL/6J from C57BL/6N were found to be segregating. This further suggests the mice sent to The Jackson Laboratory Repository were on a C57BL/6N genetic background.
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 cells where promoters driving Cre recombinase are expressed. |
Allele Name | targeted mutation 1, Feng Zhang |
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Allele Type | Targeted (Conditional ready (e.g. floxed), Reporter, Endonuclease) |
Allele Synonym(s) | Cre-dependent Cas9; Gt(ROSA)26Sortm1(CAG-xstpx-cas9,-EGFP)Fezh; Rosa26-LSL-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 cells where promoters driving Cre recombinase are expressed. |
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. |
When maintaining a live colony, these mice can be bred as homozygotes.
When using the Rosa26-LSL-Cas9 knockin mouse strain in a publication, please cite the originating article(s) and include JAX stock #024857 in your Materials and Methods section.
Facility Barrier Level Descriptions
Service/Product | Description | Price |
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Heterozygous or wildtype for Gt(ROSA)26Sor<tm1(CAG-xstpx-cas9,EGFP)Fezh>/ |
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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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