B6.Cg-Gt(ROSA)26Sor^{tm1.1(CAG-rtTA3)Slowe}/LdowJ

Stock number: 029627
Product name: Rosa26-CAGs-rtTA3 knock-in
Research areas: Neurobiology Research, Reproductive Biology Research, Research Tools

Description

The CAGs-rtTA3 (or Rosa26-CAGs-rtTA3) knock-in allele has expression of the third-generation reverse tetracycline-regulated transactivator gene (rtTA3) directed to widespread cells/tissues by Gt(ROSA)26Sor and CAG promoters. These mice are a Tet-On tool strain - allowing dox-conditional expression of TRE promoter-driven target genes.

Detailed description

The CAGs-rtTA3 (or Rosa26-CAGs-rtTA3) knock-in allele has a CAG promoter upstream of the third-generation reverse tetracycline-regulated transactivator gene (rtTA3), all inserted into the Gt(ROSA)26Sor locus. As such, widespread rtTA3 expression is observed. 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-rtTA3 to a TRE-GFP reporter line, the Rosa26-CAGs-rtTA3;TRE-GFP offspring exhibit robust GFP expression in most tissues (including both solid organs and hematopoietic cells) after the introduction of dox.
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-rtTA3 allele (Stock No. 029627) and their Rosa26-CAGs-RIK allele (a germline cre-activated rtTA3 and mKate2-expressing derivative of Stock No. 029633) 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, the shApc/Lgr5 mosaic model of shRNA-driven tumor development in the small and large intestine is created by combining the Rosa26-CAGs-LSL-rtTA3 allele (see Stock No. 029617) with the Lgr5-GFP-IRES-CreER knock-in/knock-out allele (see Stock No. 008875) and the TRE-GFP/shRNA:Apc allele (TG-APC.3374 ; Col1a1^{tm4(tetO-GFP/RNAi:Apc)Slowe}). A single tamoxifen treatment initiates cre-recombination in stem cells of the small and large intestine - inducing long-lived, rtTA3 expression in a small percentage of individual crypts and villi. Subsequent dox treatment results in shRNA-mediated Apc suppression, leading to macroscopic polyps in the small intestine and colon (4-6 seeks post treatment) that progress to large colonic polyps appearing histologically as well-differentiated tubular adenomas (12-16 weeks post treatment). Addition of the cre-inducible mutant alleles LSL-Kras^G12D allele (see Stock No. 008179) and Trp53^fl (see Stock No. 008462) results in progression to invasive carcinoma.

Mice heterozygous for the Rosa26-CAGs-rtTA3 knock-in allele are viable and fertile with no reported gross physical or behavioral abnormalities. It is the experience of The Jackson Laboratory (2019) that homozygous are viable but born at less than expected Mendelian ratios and are poor breeders.

Development

CAGs-rtTA3 (or Rosa26-CAGs-rtTA3) knock-in mice were created by the laboratory of Dr. Scott W. Lowe (Memorial Sloan Kettering Cancer Center) with a CAG promoter upstream of a reverse tetracycline-regulated transactivator (rtTA3), 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) 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. Some of the Rosa26-CAGs-LSL-rtTA3 mice (see Stock No. 029617) were bred to Zp3-Cre deleter mice (on a C57BL/6 background; Stock No. 003651) in an attempt for germline deletion of the STOP cassette. While it was removed from the targeted locus (resulting in the functional Rosa26-CAGs-rtTA3 allele), the donating investigator later found the PGK-Puro^R-SV40pA to have permanently integrated in the genome locally (and always segregated along with Rosa26-CAGs-rtTA3). Next, the Rosa26-CAGs-rtTA3 mice (Zp3-Cre removed) were transferred to Dr. Lukas E. Dow (Weill Cornell Medicine) in 2014. The Rosa26-CAGs-rtTA3 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 in 2016 as Stock No. 029627. 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, at least once during backcrossing, a heterozygous female was bred to a C57BL/6N inbred male (thus the Y chromosome of the congenic strain is of C57BL/6N origin).

Allele

Symbol: Gt(ROSA)26Sor^{tm1.1(CAG-rtTA3)Slowe}
Name: targeted mutation 1.1, Scott Lowe
MGI accession ID: MGI:5638925
Generation method: Targeted
Attributes: Transactivator
Synonyms: CAGs-rtTA3
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/6 x 129S4/SvJae)F1
Site of expression: Expression of rtTA3 is widespread.
Molecular note: The targeting construct introduced into the locus a CAG promoter, floxed stop casette and reverse tetracycline transactivator 3 (rtTA3). Cre-mediated recombination removed the stop cassette.
General note: ES cell line = C10, not C2 (Jaenisch) as reported in original reference. C10 ES cells carry Col1a1.