Overview

Also Known As: Rosa26-Cas9 knockin on B6J

These CRISPR/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.

Donating Investigator

Feng Zhang, Massachusetts Institute of Technology

Genetic overview

Genetic Background	Generation
	N5F6 (2019-03-01 00:00:00)

Gt(ROSA)26Sor^{tm1.1(CAG-cas9,-EGFP)Fezh}*

Allele Type	Gene Symbol	Gene Name
Targeted (Reporter, Endonuclease)	Gt(ROSA)26Sor	gene trap ROSA 26, Philippe Soriano

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Research Applications

Research Tools

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Base Price

Starting at:

$236.78 Domestic price for female 4-week

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Details

Detailed Description

These 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.

The 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.

Expression Data

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

Control Suggestions

000664 C57BL/6J

Additional Information

Considerations for Choosing Controls

Selected References

Platt RJ; Chen S; Zhou Y; Yim MJ; Swiech L; Kempton HR; Dahlman JE; Parnas O; Eisenhaure TM; Jovanovic M; Graham DB; Jhunjhunwala S; Heidenreich M; Xavier RJ; Langer R; Anderson DG; Hacohen N; Regev A; Feng G; Sharp PA; Zhang F. 2014. CRISPR-Cas9 Knockin Mice for Genome Editing and Cancer Modeling. Cell 159(2):440-55PubMed: 25263330 MGI: J:213550

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Genetics

Gt(ROSA)26Sor^{tm1.1(CAG-cas9,-EGFP)Fezh}*

Allele Symbol: Gt(ROSA)26Sor^{tm1.1(CAG-cas9,-EGFP)Fezh}*

Allele Name	targeted mutation 1.1, Feng Zhang
Allele Type	Targeted (Reporter, Endonuclease)
Allele Synonym(s)	targeted mutation 1.1, Feng Zhang; Gt(ROSA)26Sor^{tm1.1(CAG-cas9*,-EGFP)Fezh}
Gene Symbol and Name	Gt(ROSA)26Sor, gene trap ROSA 26, Philippe Soriano
Gene Synonym(s)	beta geo; ROSA26; R26; Gtrgeo26; Gtrosa26; Gtrgeo26; Gtrosa26; AV258896; expressed sequence AV258896; gene trap ROSA 26; gene trap ROSA b-geo 26; SETD5-AS1; Thumpd3as1
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.

Disease/Phenotype

Disease Terms

Research Areas By Genotype

This mouse can be used to support research in many areas including:

Research Tools
- CRISPR/Cas9 system
- Genetics Research
  - Mutagenesis and Transgenesis: Production of Targeted Mutations (Knockouts)

Mammalian Phenotype Terms by Genotype

The following phenotype information is associated with a similar, but not exact match to this JAX^® Mice strain

Genotype: Gt(ROSA)26Sor^{tm1.1(CAG-cas9,-EGFP)Fezh}/Gt(ROSA)26Sor^{tm1.1(CAG-cas9,-EGFP)Fezh}
involves: 129S1/Sv * 129X1/SvJ * C57BL/6 * FVB/N

normal phenotype

no abnormal phenotype detected
- mice are viable and fertile, without detectable toxicity, morphological abnormalities, tumor formation, or upregulation in DNA damage/apoptosis markers

References

Platt RJ; Chen S; Zhou Y; Yim MJ; Swiech L; Kempton HR; Dahlman JE; Parnas O; Eisenhaure TM; Jovanovic M; Graham DB; Jhunjhunwala S; Heidenreich M; Xavier RJ; Langer R; Anderson DG; Hacohen N; Regev A; Feng G; Sharp PA; Zhang F. 2014. CRISPR-Cas9 Knockin Mice for Genome Editing and Cancer Modeling. Cell 159(2):440-55PubMed: 25263330 MGI: J:213550

Additional - Gt(ROSA)26Sor^{tm1.1(CAG-cas9,-EGFP)Fezh}* related

Technical Support

Contact Technical Support

Genotyping Protocols
- Standard PCR:Generic Rosa^{em1.1(CAG-Cas9)}
- Separated PCR:Gt(ROSA)26Sor(cas9*-EGFP)
- Genotyping resources and troubleshooting
Dietary Information
- New Diet as of March 2015: Lab Diet® 5K0Q (6% fat)
Breeding Considerations

When maintaining a live colony, these mice can be bred as homozygotes. Some mice may have a white belly spot.
- Additional Breeding and Husbandry Support
Mating System
- Homozygote x Homozygote
Appearance
- occasional white belly spot
Citation
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.

Animal Health Reports

Facility Barrier Level Descriptions

AX18 (Maximum)

Pricing & Availability

Please Inquire

Domestic
International

Live Mouse

Age

Genotype

Price

weeks

Cryorecovery - Pricing

Service	Genotype	Price
	Heterozygous for Gt(ROSA)26Sor<tm1.1(CAG-cas9*,-EGFP)Fezh>

We will fulfill your order by providing at least two carriers for each strain ordered. The total number, sex, and genotypes provided will vary, although typically 8 or more animals are provided. Please check genotypes which will be recovered. While the genotypes of all animals produced will be communicated to you prior to scheduling shipment, the genotypes of animals provided may not reflect the mating scheme and genotypes described in the strain description. Animals are typically ready to ship in 11-14 weeks. If a second recovery is required to produce the minimum number of animals, then delivery time would increase to approximately 25 weeks. If we fail to produce animals of the correct genotype, you will not be charged. We cannot guarantee the reproductive success of mice shipped to your facility. If the mice are lost after the first three days (post-arrival) or do not produce progeny at your facility, a new order and fee will be necessary.

Cryorecovery to establish a Dedicated Supply for greater quantities of mice. Mice recovered can be used to establish a dedicated colony to contractually supply you mice according to your requirements. Price by quotation.

Related Products and Services

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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Terms are granted by individual review and stated on the customer invoice(s) and account statement. These transactions are payable in U.S. currency within the granted terms. Payment for services, products, shipping containers, and shipping costs that are rendered are expected within the payment terms indicated on the invoice or stated by contract. Invoices and account balances in arrears of stated terms may result in The Jackson Laboratory pursuing collection activities including but not limited to outside agencies and court filings.

The Jackson Laboratory's Genotype Promise

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.

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Licensing Information

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Also Known As: Rosa26-Cas9 knockin on B6J

Donating Investigator

Genetic overview

Research Applications

Base Price

Detailed Description

Development

Expression Data

Control Suggestions

Additional Information

Selected References

Gt(ROSA)26Sortm1.1(CAG-cas9*,-EGFP)Fezh

Allele Symbol: Gt(ROSA)26Sortm1.1(CAG-cas9*,-EGFP)Fezh

Disease Terms

Research Areas By Genotype

This mouse can be used to support research in many areas including:

Mammalian Phenotype Terms by Genotype

Genotype: Gt(ROSA)26Sortm1.1(CAG-cas9*,-EGFP)Fezh/Gt(ROSA)26Sortm1.1(CAG-cas9*,-EGFP)Fezhinvolves: 129S1/Sv * 129X1/SvJ * C57BL/6 * FVB/N

normal phenotype

References

Additional - Gt(ROSA)26Sortm1.1(CAG-cas9*,-EGFP)Fezh related

Genotyping Protocols

Dietary Information

Breeding Considerations

Mating System

Appearance

Citation

Animal Health Reports

Live Mouse

Cryorecovery - Pricing

Related Products and Services

Payment Terms and Conditions

The Jackson Laboratory's Genotype Promise

Terms of Use

Additional Use Restrictions Apply

Licensing Information

JAX® Mice, Products & Services Conditions of Use

No Warranty

Credit for PRODUCTS or SERVICES

Animal Care and Use for SERVICES

No Liability

Gt(ROSA)26Sor^{tm1.1(CAG-cas9,-EGFP)Fezh}*

Allele Symbol: Gt(ROSA)26Sor^{tm1.1(CAG-cas9,-EGFP)Fezh}*

Genotype: Gt(ROSA)26Sor^{tm1.1(CAG-cas9,-EGFP)Fezh}/Gt(ROSA)26Sor^{tm1.1(CAG-cas9,-EGFP)Fezh}
involves: 129S1/Sv * 129X1/SvJ * C57BL/6 * FVB/N

Additional - Gt(ROSA)26Sor^{tm1.1(CAG-cas9,-EGFP)Fezh}* related