Overview

Also Known As: RC::RFLTG

The RC::RFLTG triple-recombinase responsive indicator allele is designed with a rox-flanked STOP, frt-flanked STOP and loxP-flanked tdTomato::STOP all preventing transcription of eGFP. Dre- and FLP-recombinase result in high tdTomato fluorescence, and further exposure to Cre recombinase results in robust eGFP fluorescence. The RC::RFLTG allele and its derivatives are useful for fluorescent labeling of cells/tissues in embryonic or adult mice. These mice allow intersectional genetic fate mapping of different cell subpopulations defined by the overlap of three gene expression domains.

Donating Investigator

Patricia Jensen, National Institute of Environmental Health Sciences (NIEHS)

Genetic overview

Genetic Background	Generation

Gt(ROSA)26Sor^{tm1.1(CAG-tdTomato,-EGFP)Pjen}

Allele Type	Gene Symbol	Gene Name
Targeted (Conditional ready (e.g. floxed), Reporter)	Gt(ROSA)26Sor	gene trap ROSA 26, Philippe Soriano

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Research Applications

Research Tools
Neurobiology Research

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Base Price

Starting at:

$2,595.00 Domestic price Cryo Recovery

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Details

Detailed Description

The RC::RFLTG triple-recombinase responsive indicator allele has a rox-flanked STOP, frt-flanked STOP and loxP-flanked tdTomato::STOP preventing transcription of eGFP. Although under control of the endogenous Gt(ROSA)26Sor promoter/enhancer regions and the CAG hybrid promoter, widespread expression of either fluorescent protein is prevented by STOP cassettes. After removal of the first two STOP cassettes by Dre- and FLP-recombination, robust tdTomato fluorescence is expected in cells/tissues where the expression patterns of the individual promoters driving dre and Flp overlap. Further exposure to Cre recombinase removes the floxed-tdTomato::STOP, resulting in robust eGFP fluorescence.

Specifically, the donating investigator reports that RC::RFLTG mice have no tdTomato expression prior to introduction of both Dre and FLP recombinases, and no significant eGFP expression prior to introduction of Dre, FLP and Cre recombinases. Although a low level of eGFP expression is consistently present in some cell types prior to cre expression, removal of the floxed-tdTomato::STOP results in significantly greater levels of eGFP expression. The donating investigator reports that the premature eGFP fluorescence is barely visible when imaged using parameters calculated for recombinase-expressing cells. The donating investigator also reports that, for robust tdTomato expression, the order in which the rox-flanked STOP and frt-flanked STOP cassettes are removed is arbitrary.

Because both fluorescent proteins can fill the axons of neurons, the simultaneous tracing of projections from two different neuronal subpopulations is possible.

Mice homozygous for the RC::RFLTG allele are viable and fertile with no reported gross physical or behavioral abnormalities.

dre-, Flp- and/or cre-mediated removal of specific STOP cassettes within the RC::RFLTG allele results in the following derivative alleles:

i. The dual-recombinase responsive indicator allele RC::RLTG (Stock No. 026931) allows dre-inducible tdTomato fluorescence and subsequent cre-inducible eGFP fluorescence.

ii. The dual-recombinase responsive indicator allele RC::FLTG (Stock No. 026932) allows Flp-inducible tdTomato fluorescence and subsequent cre-inducible eGFP fluorescence.

iii. The single-recombinase responsive indicator allele RC::LTG has constitutive tdTomato fluorescence and allows cre-inducible eGFP fluorescence.

iv. The single-recombinase responsive indicator allele RC::RG has no tdTomato expression (tdTomato::STOP sequences removed) and allows dre-inducible eGFP fluorescence.

v. The single-recombinase responsive indicator allele RC::FG has no tdTomato expression (tdTomato::STOP sequences removed) and allows Flp-inducible eGFP fluorescence.

Development

The RC::RFLTG triple-recombinase responsive indicator allele has a rox-flanked STOP, frt-flanked STOP and loxP-flanked tdTomato::STOP upstream of the eGFP fluorescent protein, all inserted into the Gt(ROSA)26Sor locus. The specific details are below.

The pRosa-CAG-rox-FRT-loxP-tdTomato-eGFP targeting vector (pRC-RFLTG) was created in the laboratory of Dr. Patricia Jensen (National Institute of Environmental Health Sciences). The vector was designed with (from 5' to 3')

a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (from pCAGGS),
a rox-flanked His3-SV40 transcriptional STOP cassette,
a frt-flanked His3-SV40 transcriptional STOP cassette,
a loxP site,
a cDNA sequence encoding tdTomato fluorescent protein (a non-oligomerizing DsRed fluorescent protein variant with a 12 residue linker fusing two copies of the protein (tandem dimer)),
a His3-SV40 transcriptional STOP cassette,
a bovine growth hormone polyA signal,
a second loxP site,
a cDNA sequence encoding enhanced green fluorescent protein (eGFP),
a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; to enhance the mRNA transcript stability),
a bovine growth hormone polyA signal,
and an attB/attP-flanked PGK-frt-Neo-polyA cassette.

This entire targeting vector was inserted between exons 1 and 2 of the Gt(ROSA)26Sor locus via electroporation of (129S6/SvEvTac x C57BL/6)F1-derived G4 embryonic stem (ES) cells. ES cells were then transiently transfected with a PhiC31-expressing plasmid to remove the attB/attP-flanked PGK-frt-Neo-polyA cassette and replace it with the recombined attB/attP site (attL). Following ES cell injection into recipient blastocysts, chimeric males were bred to C57BL/6J females, resulting in RC::RFLTG animals.

The RC::RFLTG colony was bred with C57BL/6J wildtype mice for ten generations prior to sending to The Jackson Laboratory Repository in 2015. Upon arrival, males were used to cryopreserve sperm. To establish the living RC::RFLTG mouse colony, an aliquot of the frozen sperm was used to fertilize oocytes from C57BL/6J inbred females (Stock No. 000664).

Of note, the donating investigator reports that, at least once during backcrossing, a heterozygous female was bred to a C57BL/6J inbred male (thus the Y chromosome of the congenic strain is of C57BL/6J origin).

Expression Data

Expressed Gene	GFP, Green Fluorescent Protein,
Expressed Gene	RFP, Red Fluorescent Protein, coral
Site of Expression	GFP and tdTomato fluorescent protein will be expressed in cells where promoters driving Cre recombinase are expressed.

Control Suggestions

Approximate Controls

000664 C57BL/6J

Additional Information

Considerations for Choosing Controls

Selected References

Plummer NW; Evsyukova IY; Robertson SD; de Marchena J; Tucker CJ; Jensen P. 2015. Expanding the power of recombinase-based labeling to uncover cellular diversity. Development 142(24):4385-93PubMed: 26586220 MGI: J:226320

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Genetics

Gt(ROSA)26Sor^{tm1.1(CAG-tdTomato,-EGFP)Pjen}

Allele Symbol: Gt(ROSA)26Sor^{tm1.1(CAG-tdTomato,-EGFP)Pjen}

Allele Name	targeted mutation 1.1, Patricia Jensen
Allele Type	Targeted (Conditional ready (e.g. floxed), Reporter)
Allele Synonym(s)	Gt(ROSA)26Sor^{tm1.1(CAG-tdTomato,-EGFP)Pjen}; targeted mutation 1.1, Patricia Jensen
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	RFP, Red Fluorescent Protein, coral
Site of Expression	GFP and tdTomato fluorescent protein will be expressed in cells where promoters driving Cre recombinase are expressed.
Strain of Origin	(129S6/SvEvTac x C57BL/6NCrl)F1
Chromosome	6
Molecular Note	The vector was designed with (from 5' to 3') a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (from pCAGGS), a rox-flanked His3-SV40 transcriptional STOP cassette, a frt-flanked His3-SV40 transcriptional STOP cassette, a loxP site, a cDNA sequence encoding tdTomato fluorescent protein, a His3-SV40 transcriptional STOP cassette, a bovine growth hormone polyA signal, a second loxP site, a cDNA sequence encoding enhanced green fluorescent protein (eGFP), a woodchuck hepatitis virus post-transcriptional regulatory element, a bovine growth hormone polyA signal, and an attB/attP-flanked PGK-frt-Neo-polyA cassette. This entire targeting vector was inserted between exons 1 and 2 of the Gt(ROSA)26Sor locus. Transient transfection with a PhiC31-expressing plasmid removes the attB/attP-flanked PGK-frt-Neo-polyA cassette and replaces it with the recombined attB/attP site (attL).

Disease/Phenotype

Disease Terms

Research Areas By Genotype

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

Research Tools
- Diabetes and Obesity Research
  - loxP
- Genetics Research
  - Mutagenesis and Transgenesis
  - Tissue/Cell Markers: cell marker for bone marrow transplantation
  - Tissue/Cell Markers: transplantation marker for embryonic and adult tissue
  - Mutagenesis and Transgenesis: Cre-lox System
  - Tissue/Cell Markers
  - Tissue/Cell Markers: Cre-lox System
  - Mutagenesis and Transgenesis: Dre-Rox System
  - Tissue/Cell Markers: Dre-Rox System
- Cre-lox System
  - loxP-flanked Sequences
  - loxP-flanked Sequences: Test/Reporter
- FLP-FRT System
  - FRT-flanked Sequences: Test/Reporter
  - FRT-flanked Sequences
- Developmental Biology Research
  - transplantation marker for embryonic and adult tissue
  - Cre-lox System
- Reproductive Biology Research
  - transplantation marker for embryonic and adult tissue
  - Cre-lox System
  - Dre-Rox System
- Fluorescent Proteins
- Dre-Rox System
  - Rox-flanked Sequences
  - Rox-flanked Sequences: Test/Reporter
Neurobiology Research
- Cre-lox System
  - loxP-flanked Sequences
  - loxP-flanked Sequences: Test/Reporter
- Fluorescent protein expression in neural tissue

Mammalian Phenotype Terms by Genotype

The following phenotype relates to a compound genotype created using this strain

Genotype: Gt(ROSA)26Sor^{tm1.1(CAG-tdTomato,-EGFP)Pjen}/Gt(ROSA)26Sor^{tm1.1(CAG-tdTomato,-EGFP)Pjen}
B6.Cg-Gt(ROSA)26Sor

normal phenotype

no abnormal phenotype detected
- mice are viable and fertile

References

Plummer NW; Evsyukova IY; Robertson SD; de Marchena J; Tucker CJ; Jensen P. 2015. Expanding the power of recombinase-based labeling to uncover cellular diversity. Development 142(24):4385-93PubMed: 26586220 MGI: J:226320

Additional - Gt(ROSA)26Sor^{tm1.1(CAG-tdTomato,-EGFP)Pjen} related

Technical Support

Contact Technical Support

Genotyping Protocols
- MELT: Gt(ROSA)26Sor^{tm1.1(CAG-tdTomato,-EGFP)Pjen}
- Genotyping resources and troubleshooting
Breeding Considerations

When maintaining a live colony, homozygous mice may be bred together.
- Additional Breeding and Husbandry Support
Citation
When using the RC::RFLTG mouse strain in a publication, please cite the originating article(s) and include JAX stock #026930 in your Materials and Methods section.

Animal Health Reports

Facility Barrier Level Descriptions

Production of mice from cryopreserved embryos or sperm occurs in a maximum barrier room, G200

Pricing & Availability

Cryo Recovery

Domestic
International

Live Mouse

Age

Genotype

Price

weeks

Cryorecovery - Pricing

Service	Genotype	Price
	Heterozygous or wildtype for Gt(ROSA)26Sor<tm1.1(CAG-tdTomato,-EGFP)Pjen>

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.

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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.

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Licensing Information

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Also Known As: RC::RFLTG

Donating Investigator

Genetic overview

Research Applications

Base Price

Detailed Description

Development

Expression Data

Control Suggestions

Approximate Controls

Additional Information

Selected References

Gt(ROSA)26Sortm1.1(CAG-tdTomato,-EGFP)Pjen

Allele Symbol: Gt(ROSA)26Sortm1.1(CAG-tdTomato,-EGFP)Pjen

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-tdTomato,-EGFP)Pjen/Gt(ROSA)26Sortm1.1(CAG-tdTomato,-EGFP)PjenB6.Cg-Gt(ROSA)26Sor

normal phenotype

References

Additional - Gt(ROSA)26Sortm1.1(CAG-tdTomato,-EGFP)Pjen related

Genotyping Protocols

Breeding Considerations

Citation

Animal Health Reports

Production of mice from cryopreserved embryos or sperm occurs in a maximum barrier room, G200

Live Mouse

Cryorecovery - Pricing

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-tdTomato,-EGFP)Pjen}

Allele Symbol: Gt(ROSA)26Sor^{tm1.1(CAG-tdTomato,-EGFP)Pjen}

Genotype: Gt(ROSA)26Sor^{tm1.1(CAG-tdTomato,-EGFP)Pjen}/Gt(ROSA)26Sor^{tm1.1(CAG-tdTomato,-EGFP)Pjen}
B6.Cg-Gt(ROSA)26Sor

Additional - Gt(ROSA)26Sor^{tm1.1(CAG-tdTomato,-EGFP)Pjen} related