B6.Cg-Pax7^{tm1(cre/ERT2)Gaka}/J

Stock number: 017763
Product name: Pax7^creER
Research areas: Cell Biology Research, Internal/Organ Research, Research Tools

Description

A CreER^T2 fusion protein sequence inserted downstream of the Pax7 stop codon allows endogenous PAX7 expression in these mice while permitting specific conditional labeling, manipulation, and deletion of satellite cells.

Detailed description

An internal ribosome entry site (IRES)-CreER^T2 fusion protein was inserted 8 bp downstream of the stop codon of the paired box gene 7 (Pax7) gene. PAX7 is a marker for satellite cells, adult muscle stem cells required for muscle regeneration after injury. Unlike B6;129-Pax7^{tm2.1(cre/ERT2)Fan}/J mice (Stock No. 012476), in which PAX7 expression is abolished, PAX7 is expressed and functional in these mice. Homozygotes are viable and fertile. Cre-ER^T2 fusion gene activity is inducible and only observed following tamoxifen administration. When these mice are bred with mice containing loxP-flanked sequence, tamoxifen-inducible, Cre-mediated recombination will result in deletion of the floxed sequences in the Pax7-expressing cells of the offspring. For example when bred to B6;129-Gt(ROSA)26Sor^tm1(DTA)Mrc/J mice (Stock No. 010527), tamoxifen induction results in diphtheria toxin A expression in satellite cells, and subsequent ablation of satellite cells. These double mutant mice exhibit complete loss of muscle regeneration, misregulation of fibroblasts, and an increase of muscle connective tissue.

The Cre-ER^T2 fusion protein consists of Cre recombinase fused to a triple mutant form of the human estrogen receptor which does not bind its natural ligand (17β-estradiol) at physiological concentrations but will bind the synthetic estrogen receptor ligands 4-hydroxytamoxifen (OHT or tamoxifen) and, with lesser sensitivity, ICI 182780. Restricted to the cytoplasm, Cre-ER^T2 can only gain access to the nuclear compartment after exposure to tamoxifen. To counteract the mixed estrogen agonist effects of tamoxifen injections, which can result in late fetal abortions in pregnant mice, progesterone may be coadministered.

Development

A targeting vector was designed to insert an internal ribosome entry site (IRES)-CreER^T2 fusion protein, followed by a frt-flanked neomycin (neo) selection cassette, 8bp downstream of the endogenous stop codon of the paired box gene 7 (Pax7) gene. This construct was electroporated into (129S6/SvEvTac x C57BL/6NCr)F1-derived G4 embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts and chimeric males were bred with C57BL/6 females. The resulting mice were bred to 129S4/SvJaeSor-Gt(ROSA)26Sor^tm1(FLP1)Dym/J mice (Stock No. 003946) to remove the neo cassette. These Pax7^cre/ERT2mice were backcrossed to C57BL/6J for at least 8 generations. Upon arrival, mice were bred to C57BL/6J (Stock No. 000664) for at least one generation.

Allele

Symbol: Pax7^{tm1(cre/ERT2)Gaka}
Name: targeted mutation 1, Gabrielle Kardon
MGI accession ID: MGI:5141477
Generation method: Targeted
Attributes: Recombinase-expressing; Inducible
Marker symbol: Pax7
Marker name: paired box 7
Chromosome: 4
Strain of origin: (129S6/SvEvTac x C57BL/6NCrl)F1
Expressed genes: cre/ERT2,Cre recombinase and estrogen receptor 1 (human) fusion gene,
Site of expression: When these mice are bred with mice containing loxP-flanked sequence, tamoxifen-inducible, Cre-mediated recombination will result in deletion of the floxed sequences in Pax7-expressing satellite cells, and subsequent ablation of satellite cells in the offspring.
Molecular note: An IRES-cre/ERT2 and an FRT flanked neomycin selection cassette were inserted 8 bp after the endogenous stop codon. The selection cassette was subsequently removed by crossing to a Gt(ROSA)26Sor^tm1(FLP1)Dym mouse line. Endogenous gene function is preserved.