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

Stock number: 026930
Product name: RC::RFLTG
Research areas: Neurobiology Research, Research Tools

Description

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.

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).

Allele

Symbol: Gt(ROSA)26Sor^{tm1.1(CAG-tdTomato,-EGFP)Pjen}
Name: targeted mutation 1.1, Patricia Jensen
MGI accession ID: MGI:5617961
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter
Marker symbol: Gt(ROSA)26Sor
Marker name: gene trap ROSA 26, Philippe Soriano
Chromosome: 6
Strain of origin: (129S6/SvEvTac x C57BL/6NCrl)F1
Expressed genes: RFP,Red Fluorescent Protein,coral; GFP,Green Fluorescent Protein,
Site of expression: GFP and tdTomato fluorescent protein will be expressed in cells where promoters driving Cre recombinase are expressed.
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).