B6.129S6-Gt(ROSA)26Sor^{tm12(CAG-Venus*,-TagRFP*)Rkuhn}/MurrJ

Stock number: 034033
Product name: traffic light reporter (TLR2) , R26-GFP_KI-TLR2
Strain synonyms: TLR-2
Research areas: Cancer Research, Neurobiology Research, Research Tools

Description

R26-GFP_KI-TLR2 ("traffic light reporter") knock-in mice have a CAG promoter controlling expression of Venus (GFP) and TagRFP inserted in the Gt(ROSA)26Sor locus and is a reporter for DNA repair pathways.

Of note: the TLR2 allele also is distributed on a mixed C57BL/6 and 129S6 background - Stock No. 032672.

Detailed description

This R26-GFP_KI-TLR2 "traffic light reporter" knock-in line has a CAG promoter controlling expression of a mutant (non-functional) Venus (GFP) and TagRFP inserted into the endogenous Gt(ROSA)26Sor locus. The strain is designed to express GFP when a double-strand break occurs and is repaired by homology-directed repair (HDR), similarly RFP is expressed when a double-strand break occurs and is repaired by nonhomologous end-joining (NHEJ). Homozygotes are viable and fertile. As the mice are characterized, we will modify the strain description and add phenotype data.

Of note: the TLR2 allele also is distributed on a mixed C57BL/6 and 129S6 background - Stock No. 032672.

The TLR2 allele is similar to the TLR-7 allele (Stock No. 034956), but TLR-7 is designed with a shorter interruption in Venus that facilitates oligonucleotide-mediated HDR for activation of Venus.

Development

The R26-GFP_KI-TLR2 ("traffic light reporter") targeting vector was designed to insert the following into the Gt(ROSA)26Sor locus: FRT site, PGK promoter, two recombined attP/B sites, neomycin resistance cassette, polyA, FRT site and the TLR reporter construct ("TLR2") containing a CAG promoter Venus*-P2A-TagRFP-bovine hGH polyA, in a reading frame that is shifted by 2 bp (+3) [see Chu et al. Nat Biotechnol. 2015 33:543 - Fig. 1B]. The Venus* used here is a non-functional Venus having codons 117-152 replaced with 52bp of mouse Gt(ROSA)26Sor and 56bp of mouse Rab38 (RAB38, member RAS oncogene family sequence). The targeting construct was electroporated into (C57BL/6J x 129S6/SvEvTac)F1-derived IDG3.2 embryonic stem (ES) cells. Correctly targeted ES cells were injected into blastocysts. The resulting chimeric animals were tested for germline transmission. The mice were then backcrossed to C57BL/6J for 3 generations (during which, the Y chromosome may not have been fixed to the C57BL/6J genetic background). Sperm from homozygous mice that were a mix of about 12.5% 129S6/SvEvTac and 87.5% C57BL/6J were imported. That sperm was recovered on the C57BL/6J background to establish our live colony as Stock No. 032672. Upon arrival, some mice were further backcrossed to C57BL/6J using a marker-assisted, speed congenic protocol. The resulting C57BL/6J congenic strain is Stock No. 034033.

Allele

Symbol: Gt(ROSA)26Sor^{tm12(CAG-Venus*,-TagRFP*)Rkuhn}
Name: targeted mutation 12, Ralf Kuhn
MGI accession ID: MGI:6324910
Generation method: Targeted
Attributes: Reporter
Synonyms: Gt(ROSA)26Sor^{tm1(CAG-Venus*,TagRFP*)Rkuhn}
Marker symbol: Gt(ROSA)26Sor
Marker name: gene trap ROSA 26, Philippe Soriano
Marker synonyms: beta geo; Gtrgeo26; Gtrosa26; R26; ROSA26; SETD5-AS1; Thumpd3as1
Chromosome: 6
Strain of origin: (C57BL/6J x 129S6/SvEvTac)F1
Site of expression: When a double-strand break is generated at the target region (nuclease cleavage site) in Gt(ROSA)26Sor, the traffic light reporter is designed to express GFP when the double-strand break is repaired by homology-directed repair (HDR), and RFP when the double-strand break is repaired by nonhomologous end-joining (NHEJ).
Molecular note: The R26-GFP_KI-TLR2 ("traffic light reporter") targeting vector containing Venus*-P2A-TagRFP-bovine hGH polyA, in a reading frame that is shifted by 2 bp (+3), under the control of a CAG promoter, and with an upstream PGK-neo cassette, two FRT sites and 2 recombined attP/B sites, was inserted into the Gt(ROSA)26Sor locus. The non-functional Venus in the TLR vector has codons 117-152 replaced with 52bp of mouse Gt(ROSA)26Sor and 56bp of mouse Rab38 (RAB38, member RAS oncogene family sequence).