B6;129S-Igs7^{tm166(tetO-EGFP*,CAG-tTA2)Hze}/XwyJ

Stock number: 035404
Product name: TIGRE-MORF (Ai166)
Research areas: Cancer Research, Neurobiology Research, Research Tools

Description

TIGRE-MORF(Ai166) is one of a set of a Cre reporter tool strains (also including MORF1 and MORF3) that use mononucleotide repeat frameshift (MORF) as a translation switch for cell labeling in vivo. TIGER-MORF (Ai166) mice express a cre-dependent tetracycline-activated EGFP fluorescent protein that is proceeded by a polyguanine repeat (G₂₂) MORF switch. Exposure to Cre recombinase removes both STOP cassettes unblocking EGFP fluorescence and tTA2 expression and in the presence of spontaneous frameshift the EGFP reporter provides sparse and stochastic labeling of neural cells. In addition, reporter expression may be diminished/eliminated by doxycycline. TIGER-MORF(Ai166) can be used for high level direct fluorescent sparse labeling of Cre+ neuronal and glial cells.

Detailed description

TIGRE-MORF(Ai166) is one of a set of a Cre reporter tool strains that use mononucleotide repeat frameshift (MORF) as a translation switch for sparse cell labeling in vivo. Mononucleotide repeats naturally exist in the genomes of many organisms, including mammals, and are susceptible to stochastic frameshift mutations during DNA replication or repair. TIGRE-MORF(Ai166) is designed to have a cre-dependent, tetracycline-activated tTA2-TRE transcriptional cassette combined with a Kozak consensus start site (for strong initiation of translation), two Myc-tags, a polyguanine repeat (G₂₂) MORF translation switch, and a membrane-bound (farnesylated) EGFP fluorescent reporter inserted into the Igs7 locus (TIGRE; an intergenic region on mouse chromosome 9 that allows reporter expression to be tightly regulated). Background expression of EGFP in TIGR-MORF mice is not detected.

When bred to mice that express Cre recombinase, the resulting offspring will have both STOP cassettes deleted in the cre-expressing tissue(s), unblocking EGFP fluorescence and tTA2 expression. However, due to the presence of the mononucleotide repeat, the EGFP reporter remains out of frame and is not expressed until a spontaneous frameshift occurs. Once the reporter is in-frame a small and random subset of Cre+ cells are labeled at a labeling frequency of 1-5%. Homozygous TIGRE-MORF(Ai166) mice are viable and fertile. Of the three MORF strains (MORF1, MORF3 and TIGRE-MORF) TIGRE-MORF(Ai166) has the strongest labeling strength with direct fluorescent imaging.

Additional MORF strains available are the cre-dependent MORF3, which uses a polycytosine MORF repeat and a multivalent immunoreporter (Stock No. 035403) and MORF1 (Stock No. 035400), which uses a cre-dependent polycytosine MORF repeat with a farnesylated mNeonGreen reporter.

Development

The TIGRE-MORF(Ai166) reporter contains a cre-dependent, tetracycline-activated tTA2-TRE transcriptional cassette combined with a Kozak consensus start site (for strong initiation of translation), two Myc-tags, an optimized G₂₂-repeat MORF translation switch, and a farnesylated EGFP fluorescent reporter. The farnesylation sequence (CAAX domain from Ras) targets the reporter to membranous structures including neuronal dendrites and axons.

Specifically, the final vector contains from 5' to 3': FRT3, 2x Ins, TRE promoter, loxP-flanked STOP sequence, a Kozak consensus start site (for strong initiation of translation), two Myc-tags, an optimized G₂₂-repeat MORF switch, a farnesylated EGFP fluorescent reporter (EGFP-F), WPRE, pA, 2X Ins, CAG promoter, lox2272-flanked STOP sequence, tTA2, WPRE, pA and an attB-PGK-5'hygro-SD-frt5-SA-3'hygro-SV40pA-attP, inserted into the T1 TIGRE locus (an intergenic region on chromosome 9 [between AB124611 and Carm1] determined to have low basal transcriptional activity and allow high cre and/or Tet inducibility). (129S6/SvEvTac x C57BL/6)F1-derived G4 embryonic stem cells previously targeted with FRT3::attB::PGK-neoR-polyA::FRT5::splice acceptor::3'hygro cassette::SV40 polyA:attP in TIGRE, were re-transfected with the replacement vector and Flp recombinase vector for recombinase-mediated cassette exchange (RCME). Chimeric mice were bred with C57BL/6J-congenic PhiC31 mice (see Stock No. 007743). Genotyping at The Jackson Laboratory indicates that the hygromycin cassette is still present. Mice were backcrossed to C57BL/6J mice for at least 6 generations by the donating laboratory (see SNP note below). Upon arrival, mice were bred to C57BL/6J for at least 1 generation to establish the colony.

In 2021, 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 males sent to The Jackson Laboratory Repository. Three of the 43 markers throughout the genome were segregating with 129, suggesting an incomplete backcross.

Allele

Symbol: Igs7^{tm166(tetO-EGFP*,CAG-tTA2)Hze}
Name: targeted mutation 166, Hongkui Zeng
MGI accession ID: MGI:6441963
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter; Inducible; Transactivator
Synonyms: Ai166; TIGRE-MORF
Marker symbol: Igs7
Marker name: intergenic site 7
Marker synonyms: Iis7; TIGRE
Chromosome: 9
Strain of origin: (129S6/SvEvTac x C57BL/6NCrl)F1
Site of expression: tetO-inducible of EGFP and cre-inducible widespread expression of tTA2 protein

Molecular note: The reporter contains a cre-dependent, tetracycline-activated tTA2-TRE transcriptional cassette combined with a Kozak consensus start site (for strong initiation of translation), two Myc-tags, an optimized G)22(-repeat MORF translation switch, and a farnesylated EGFP fluorescent reporter. The farnesylation sequence (CAAX domain from Ras) targets the reporter to membranous structures including neuronal dendrites and axons. Specifically, the final vector contains from 5' to 3': FRT2, 2x Ins, TRE promoter, loxP-flanked STOP sequence, a Kozak consensus start site (for strong initiation of translation),two Myc-tags, an optimized G)22(-repeat MORF switch, a farnesylated EGFP fluorescent reporter (EGFP-F), WPRE, pA, 2X Ins, CAG promoter, lox2272-flanked STOP sequence, tTA2, WPRE, pA and an attB-PGK-5';hygro-SD-frt5-SA 3';hygro-SV40pA-attPan FRT site, inserted into the T1 TIGRE locus.