B6;129-Gt(ROSA)26Sor^{tm1(rtTA*M2)Jae} Col1a1^{tm1(tetO-cre)Haho}/J

Stock number: 021025
Product name: R26-M2rtTA::TetOP-Cre
Research areas: Cancer Research, Cell Biology Research, Neurobiology Research, Research Tools

Description

These double mutant R26-M2rtTA::TetOP-Cre mice utilize the rtTA/tetO (tet-on/TRE) system to allow highly-efficient, doxycycline-inducible Cre recombinase expression in hematopoietic stem cells. These mice harbor the R26^M2rtTA targeted mutation (an optimized form of reverse tetracycline controlled transactivator [rtTA-M2] downstream of the Gt(ROSA)26Sor promoter) and the Col1a1^TetOP-Cre targeted mutation (a tetracycline responsive element-driven Cre recombinase targeted to the collagen type I alpha 1 locus).

Detailed description

R26-M2rtTA::TetOP-Cre mice harbor the Rosa26-rtTA targeted mutation and the Col1a1^TetOP-Cre targeted mutation. Rosa26-rtTA has an optimized form of reverse tetracycline controlled transactivator (rtTA-M2) downstream of the Gt(ROSA)26Sor promoter. Col1a1^TetOP-Cre contains a tetracycline responsive element-driven Cre recombinase targeted to the collagen type I alpha 1 locus.
Mice homozygous for both targeted mutations (homozygous R26-M2rtTA::TetOP-Cre mice) or heterozygous for both targeted mutations (heterozygous R26-M2rtTA::TetOP-Cre mice) are viable and fertile. Following administration of the tetracycline analog doxycycline (dox), the donating investigator reports sufficient Cre recombinase activity is observed for both homozygous R26-M2rtTA::TetOP-Cre mice and heterozygous R26-M2rtTA::TetOP-Cre mice. Specifically, homozygous R26-M2rtTA::TetOP-Cre mice exhibit Cre recombinase activity in hematopoietic stem cells at high efficiency (close to 100% for floxed alleles that face no selection in hematopoietic stem cells upon recombination); and dox-inducible Cre recombinase activity in adult tissues is reported at ~30-50% efficiency. In the absence of dox, heterozygous R26-M2rtTA::TetOP-Cre mice exhibit no Cre recombinase expression from the Col1a1 locus. Homozygous R26-M2rtTA::TetOP-Cre mice have a low degree of Cre recombinase expression prior to dox administration (causing low levels of recombination with some floxed alleles; less than 10% germline excision occurs). In vitro, a minor reduction in the rate of hematopoietic progenitor colony growth is observed for homozygous R26-M2rtTA::TetOP-Cre mice, but not heterozygous R26-M2rtTA::TetOP-Cre mice.

Development

The R26-M2rtTA targeted mutation was created by Dr. Rudolf Jaenisch (Whitehead Institute, MIT) with an optimized form of reverse tetracycline controlled transactivator (rtTA-M2) followed by a β-globin intron, polyA signal and PGK-puromycin cassette, all inserted downstream of the endogenous promoter at the Gt(ROSA)26Sor locus. The targeting vector was electroporated into (C57BL/6 x 129S4Sv/Jae)F1-derived V6.5 embryonic stem (ES) cells. Next, these targeted ES cells (termed V19 ES cells) were retargeted to insert an frt-flanked PGK-Neo into the 3' UTR of the Col1a1 locus. Correctly targeted ES cells were selected. Next, Dr. Hanno R. Hock (Massachusetts General Hospital) replaced the frt-flanked PGK-Neo in the 3' UTR of the Col1a1 locus with a TetOP-Cre sequence. This was performed by injecting a "TetOP-Cre flip-in plasmid" (containing a splice acceptor-polyA sequence and the tetracycline operator elements fused to a CMV minimal promoter [TRE or tetO] upstream of a Cre recombinase cassette [from pTurbo-cre; Genbank AF334827.1]), along with a Flpe-expressing plasmid (to facilitate TetOP-Cre recombination into the 3' UTR of the Col1a1 locus). The resulting ES cells, now targeted with rtTA-M2 in the Gt(ROSA)26Sor locus and TetOP-Cre in the Col1a1 locus (R26-M2rtTA::TetOP-Cre) were injected into B6D2F1 tetraploid blastocysts. The double mutant chimeric males were bred with C57BL/6J females to generate the colony. Double mutant pups on the resulting C57BL/6;129 mixed genetic background were bred together for several generations, and then sent to The Jackson Laboratory Repository in 2014. Upon arrival, mice were bred with C57BL/6J inbred mice (Stock No. 000664) for at least one generation.

Allele

Symbol: Col1a1^{tm1(tetO-cre)Haho}
Name: targeted mutation 1, Hanno Hock
MGI accession ID: MGI:5558024
Generation method: Targeted
Attributes: Recombinase-expressing; Inducible
Marker symbol: Col1a1
Marker name: collagen, type I, alpha 1
Chromosome: 11
Strain of origin: (C57BL/6 x 129S4/SvJae)F1
Expressed genes: cre,cre recombinase,bacteriophage P1
Site of expression: When combined with a reverse tetracycline controlled transactivator, cre recombinase expression can be induced in hematopoietic stem cells by doxycycline administration.
Molecular note: A "TetOP-Cre flip-in plasmid" (containing a splice acceptor-polyA sequence and the tetracycline operator elements (tet)) fused to a CMV minimal promoter upstream of a cre recombinase cassette) was used to treat ES cells containing an FRT-PGK-neo-hygro-FRT cassette inserted into the 3' UTR of the Col1a1 locus (Col1a1^{tm13(neo/hygro)Jae}). The "flip-in" vector was coinjected with a Flpe-encoding plasmid so that flippase-mediated recombination inserted the tetO-cre sequence into the Col1a1 3' UTR. The Col1a1^{tm13(neo/hygro)Jae} ES cells also contain the Gt(ROSA)26Sor^{tm1(rtTA*M2)Jae} allele. The twice-targeted ES cells were used to generate chimeras and germline animals. By treating this mouse line with doxycycline, rtTA*M2 binds the TetOP promoter and cre is expressed.

Allele

Symbol: Gt(ROSA)26Sor^{tm1(rtTA*M2)Jae}
Name: targeted mutation 1, Rudolf Jaenisch
MGI accession ID: MGI:3702294
Generation method: Targeted
Attributes: Transactivator
Marker symbol: Gt(ROSA)26Sor
Marker name: gene trap ROSA 26, Philippe Soriano
Chromosome: 6
Strain of origin: (C57BL/6 x 129S4/SvJae)F1
Expressed genes: rtTA,reverse tetracycline-controlled transactivator,E. coli
Site of expression: Expresses an optimized rtTA protein (rtTA-M2). Inducible target gene expression is detected in liver, bone marrow, stomach, intestine, and skin, with lower levels in the heart, lungs, kidney, spleen, and thymus; no expression is detected in the brain and testes.
Molecular note: An optimized form of reverse tetracycline controlled transactivator (rtTA-M2) was inserted downstream of the Gt(ROSA)26Sor promoter and was followed by a PGK-puro selection cassette. This mutant form of rtTA termed M2 has five amino acid substitutions in the tetR moiety of tTA: S12G, E19G, A56P, D148E and H179R. This mutated form of transactivator protein has increased doxycycline sensitivity. Mice have widespread expression of the rtTA-M2 protein.
General note: This mutation was created during the generation of mutant ES cell line KH2, which also contains a frt docking site downstream of the endogenous Col1a1 locus on Chr 11 (Col1a1). The two mutations have subsequently been bred apart.