B6.Cg-Gt(ROSA)26Sor^{tm2(CAG-rtTA3,-mKate2)Slowe}/J

Stock number: 029633
Product name: Rosa26-CAGs-LSL-RIK knock-in
Research areas: Cancer Research, Neurobiology Research, Reproductive Biology Research, Research Tools

Description

CAGs-LSL-RIK (or Rosa26-CAGs-LSL-RIK) knock-in has a CAG promoter and loxP-flanked STOP cassette upstream of the third-generation reverse tetracycline-regulated transactivator gene (rtTA3), IRES sequence and the monomeric far-red fluorescent protein mKate2, all inserted into the Gt(ROSA)26Sor locus. These mice are a Cre recombinase-dependent, Tet-On and fluorescent tool strain - following removal of the floxed-STOP cassette, they allow dox-conditional expression of TRE promoter-driven target genes in conjunction with mKate2 fluorescence.

Detailed description

The CAGs-LSL-RIK (or Rosa26-CAGs-LSL-RIK) knock-in allele has a CAG promoter and loxP-flanked STOP cassette upstream of the third-generation reverse tetracycline-regulated transactivator gene (rtTA3), IRES sequence and the monomeric far-red fluorescent protein mKate2, all inserted into the Gt(ROSA)26Sor locus. In the absence of Cre recombinase, a floxed STOP cassette prevents expression of rtTA3 and mKate2. Following cre-mediated deletion of the floxed-STOP cassette, mKate2 fluorescence is observed - and subsequent expression of a target gene under control of a tetracycline-responsive promoter element (TRE; or tetO) can be induced/regulated by tetracycline or its analog doxycycline (dox).

For example, breeding Rosa26-CAGs-LSL-RIK to a germline Cre-deleter results in Rosa26-CAGs-RIK mice with widespread, constitutive expression of the two individual proteins - rtTA3 and mKate2. When those Rosa26-CAGs-RIK mice are bred to a TRE-GFP reporter line, the Rosa26-CAGs-RIK;TRE-GFP offspring exhibit robust GFP expression in most tissues (including both solid organs and hematopoietic cells) after the introduction of dox. As expected, Rosa26-CAGs-RIK mice show uniform expression of mKate2 (fluorescence and/or immunostaining) in all tissues examined. However, some Rosa26-CAGs-RIK;TRE-GFP splenic B cells showed poor GFP reporter (despite comparable rtTA3 expression [mRNA and protein] in GFP^hi and GFP^dim cells).
Specifically, the donating investigator's publication (Dow et al. 2014 PLoS One 9:e95236) uses a TRE-GFP reporter line to compare their Rosa26-CAGs-RIK allele and their Rosa26-CAGs-rtTA3 allele (Stock No. 029627) to the Rosa26-rtTA^M2 allele (Stock No. 006965). Whereas all three double mutant lines (Rosa26-CAGs-rtTA3;TRE-GFP, Rosa26-CAGs-RIK;TRE-GFP and Rosa26-rtTA*M2;TRE-GFP) exhibit high-level GFP induction in T cells and intestine, the Rosa26-CAGs-rtTA3;TRE-GFP and Rosa26-CAGs-RIK;TRE-GFP lines show significantly higher GFP induction in skin, liver, kidney and pancreas. In addition, immunofluorescent analysis of Rosa26-CAGs-rtTA3;TRE-GFP and Rosa26-CAGs-RIK;TRE-GFP revealed uniform and consistent GFP staining in multiple cell types in each organ analyzed, whereas Rosa26-rtTA*M2;TRE-GFP often showed absent or patchy expression in organs including pancreas and liver. Importantly, both Rosa26-CAGs-rtTA3;TRE-GFP and Rosa26-CAGs-RIK;TRE-GFP showed almost identical GFP reporter expression - indicating that the presence of mKate2 in the Rosa26-CAGs-RIK allele did not alter rtTA3 level. Overall, in combination with an appropriate cre-expressing mouse line, the Rosa26-CAGs-LSL-rtTA3 and Rosa26-CAGs-LSL-RIK strains can provide robust, tissue and/or cell-type specific TRE-mediated gene expression.

In addition, a pancreatic cancer model is created by combining the Rosa26-CAGs-LSL-RIK allele with the Pdx1-Cre transgene (see Stock No. 014647), the cre-inducible LSL-Kras^G12D allele (see Stock No. 008179) and the TRE-GFP/shRNA:Luc allele (TGRen.713 ; Col1a1^{tm7(tetO-GFP/RNAi:rluc)Slowe}). Along with dox treatment for three months, mosaic cre-recombination in pancreas leads to mKate2 and GFP expression, as well as the expected Kras^G12D phenotype: normal acinar tissue and the development of acinar-to-ductal metaplasia and early PanIN lesions.
Similarly, the HER2-driven breast cancer model MMTV-neu^V664E (see Stock No. 005038) can be used with the Rosa26-CAGs-LSL-RIK allele, the WAP-Cre transgene (see Stock Nos. 003552/008735) and the TRE-GFP/shRNA:Luc allele (TGRen.713 ; Col1a1^{tm7(tetO-GFP/RNAi:rluc)Slowe}). Along with dox treatment, cre-recombination in luminal epithelia leads to mKate2 and GFP expression, as well as the expected MMTV-neu^V664E-driven tumors - some of which were mKate2+ and GFP+ (where MMTV and WAP expression overlaps).
Taken together, these studies show the Rosa26-CAGs-LSL-RIK allele may be used effectively as the Tet-On driver in combination with established cancer models to express inducible shRNAs.

Mice heterozygous or homozygous for the Rosa26-CAGs-LSL-RIK knock-in allele are viable and fertile with no reported gross physical or behavioral abnormalities.

Development

CAGs-LSL-RIK (or Rosa26-CAGs-LSL-RIK) knock-in mice were created by the laboratory of Dr. Scott W. Lowe (Memorial Sloan Kettering Cancer Center) with a CAG promoter and loxP-flanked STOP cassette upstream of a reverse tetracycline-regulated transactivator gene (rtTA3), IRES sequence and mKate2 fluorescent protein, all inserted into the Gt(ROSA)26Sor locus. The specific details are below.

The targeting vector was designed with (from 5' to 3') a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (from pCAGGS), a loxP-flanked STOP cassette (LSL; this one containing a reverse-oriented PGK-Puro^R and SV40 polyA signal), a sequence encoding the third-generation reverse tetracycline-regulated transactivator gene (rtTA3), an internal ribosome entry site sequence (IRES; allows translation initiation in the middle of an mRNA sequence), a sequence encoding the monomeric far-red fluorescent protein mKate2 (optimized for high-brightness, photostability, pH resistance and low-toxicity) and a beta-globin polyA signal. The entire targeting vector was inserted between exons 1 and 2 of the Gt(ROSA)26Sor locus via electroporation into (C57BL/6 x 129S4Sv/Jae)F1-derived C10 embryonic stem (ES) cells. Correctly targeted ES cells were selected and mice were generated by tetraploid embryo complementation. Breeding to C57BL/6N mice led to germline transmission. The Rosa26-CAGs-LSL-RIK colony was backcrossed to C57BL/6N wildtype mice for a total of ten generations prior to sending males with black coat color to The Jackson Laboratory Repository. Upon arrival, males were used to cryopreserve sperm. To establish a living mouse colony, an aliquot of the frozen sperm may be used to fertilize oocytes from C57BL/6NJ inbred females (Stock No. 005304).

Of note, the donating investigator reports that the Y chromosome may not have been fixed to the C57BL/6N background during backcrossing.

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 males sent to The Jackson Laboratory Repository. Two of the 43 markers throughout the genome were segregating with 129 on Chromosome 12. Also, 1 of the 5 markers that determine C57BL/6J from C57BL/6N was found to be segregating on Chromosome 6. These data suggest the mice sent to The Jackson Laboratory Repository were on a mixed C57BL/6N;C57BL/6J genetic background.

Allele

Symbol: Gt(ROSA)26Sor^{tm2(CAG-rtTA3,-mKate2)Slowe}
Name: targeted mutation 2, Scott Lowe
MGI accession ID: MGI:5638918
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter; Transactivator
Synonyms: R26Sor^{tm2(CAGs-LSL-RIK)Slo}
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 129S4/SvJae)F1
Expressed genes: rtTA,reverse tetracycline-controlled transactivator,E. coli
Site of expression: When these mutant mice are bred to mice that express Cre recombinase, the resulting offspring will express rtTA3 in cre-expressing tissues with subsequent expression of RFP (mKate2) under control of a tetracycline-responsive promoter element (TRE; or tetO) that can be induced/regulated by tetracycline or its analog doxycycline (dox).
Molecular note: The targeting construct introduced into the locus a CAG promoter, floxed stop cassette, reverse tetracycline transactivator 3 (rtTA3), IRES and a far red reporter gene (mKate2).
General note: ES cell line = C10, not C2 (Jaenisch) as reported by original reference. C10 ES cells carry Col1a1.