B6.Cg-Hprt^{tm353(Ple274-icre/ERT2)Ems}/Mmjax

Stock number: 023698

Description

Ple274-icre/ERT2;mEMS5758 mice have the Ple274-icre/ERT2 transgene targeted as a single copy "knock-in" into the upstream region of the hypoxanthine guanine phosphoribosyl transferase (Hprt) locus on the X Chromosome. This is designed to allow the promoter/enhancer/regulatory regions of the human corticotropin releasing hormone (CRH) locus to direct expression of the tamoxifen-inducible, improved Cre recombinase (icre/ERT2).

Detailed description

Ple274-icre/ERT2;mEMS5758 mice have the Ple274-icre/ERT2 transgene targeted as a single copy "knock-in" into the upstream region of the hypoxanthine guanine phosphoribosyl transferase (Hprt) locus on the X Chromosome. Heterozygous females and hemizygous males are viable and fertile, with the promoter/enhancer/regulatory regions of the human corticotropin releasing hormone (CRH) locus directing expression of the tamoxifen-inducible, improved Cre recombinase (icre/ERT2). The donating investigator reports RT PCR expression as "Faint Positive (all brain regions tested)." The phenotype of homozygous mice has not been evaluated to date (January 2014).

The iCre/ER^T2 fusion protein used here consists of a codon-improved Cre recombinase fused to a G400V/M543A/L544A 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, iCre/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

Ple274-icre/ERT2;mEMS5758 mice were created by the CanEuCre and Pleiades Promoter Project (Centre for Molecular Medicine and Therapeutics, University of British Columbia). These mice have the Ple274-icre/ERT2 transgene targeted as a single copy "knock-in" into the upstream region of the hypoxanthine guanine phosphoribosyl transferase (Hprt) locus on the X Chromosome. As such, the promoter/enhancer/regulatory regions of the human corticotropin releasing hormone (CRH) locus are designed to direct expression of the tamoxifen-inducible, improved Cre recombinase (icre/ERT2). More specific details are described below.

The RP11-1006.F7 human bacterial artificial chromosome (BAC) was obtained and retrofitted to contain a human HPRT complementary sequence (containing exon 1, intron 1, exon 2 and part of intron 2), a mouse 3' Hprt homology arm, an I-Sce linearization cut site, and a mouse 5' Hprt homology arm; all inserted into the sacB gene in the BAC vector backbone (thus disrupting the sacB gene and removing the loxP site in the vector backbone). Next, an iCre/ERT2 fusion gene and an Ffrt-flanked kanamycin cassette were inserted directly at the ATG of the human corticotropin releasing hormone (CRH) locus. The kanamycin cassette was then permanently removed via transient transfection of a FLP recombinase plasmid; creating the Ple274-icre/ERT2 BAC (bEMS192) used as the source of the Ple274-icre/ERT2 transgene. This transgene was targeted as a single copy knock-in to the Hprt^b-m3 mutant locus on the X Chromosome via electroporation into mEMS4857.02 embryonic stem cells (described below). The resulting chimeric males (founder line mEMS5758) were bred to C57BL/6J females to establish the mutant colony. The donating investigator reports that Ple274-icre/ERT2;mEMS5758 mutant animals were backcrossed to C57BL/6J mice for 3 generations, and then sperm was frozen from black hemizygous males at the University of British Columbia. In 2013-2014, frozen sperm was sent to the Mutant Mouse Regional Resource Center node at The Jackson Laboratory (MMRRC-JAX). When rederiving the live colony, an aliquot of frozen sperm is used to fertilize oocytes from C57BL/6J female mice (Stock No. 000664); the resulting obligate heterozygous females are bred at least one generation to C57BL/6J mice to establish the live colony.

mEMS4857.02 embryonic stem (ES) cells are a male ES cell line made from an NE4.75F4 blastocyst from C57BL/6NTac-A^w-j/A^w-j Hprt^b-m3/Y mice (created by four generations of breeding C57BL/6NTac-A^w-J together with C57BL/6NTac-Hprt^b-m3). The parental lines were individually made by backcrossing C57BL/6J-A^w-J/J (Stock No. 000051) four generations onto C57BL/6NTac, and backcrossing B6.129P2-Hprt^b-m3/J (Stock No. 002171) six generations onto C57BL/6NTac, respectively.

Allele

Symbol: Hprt1^{tm353(Ple274-icre/ERT2)Ems}
Name: targeted mutation 353, Elizabeth M Simpson
MGI accession ID: MGI:5568222
Generation method: Targeted
Attributes: Recombinase-expressing; Inducible
Marker symbol: Hprt1
Marker name: hypoxanthine phosphoribosyltransferase 1
Chromosome: X
Strain of origin: B6N.Cg-A^w-J Hprt^b-m3
Expressed genes: cre/ERT2,Cre recombinase and estrogen receptor 1 (human) fusion gene,
Molecular note: The transgene consists of a RP11-1006.F7 human bacterial artificial chromosome (BAC) retrofitted to contain a human HPRT complementary sequence (containing exon 1, intron 1, exon 2 and part of intron 2), a mouse 3' Hprt homology arm, an I-Sce linearization cut site, and a mouse 5' Hprt homology arm; all inserted into the sacB gene in the BAC vector. Next, an iCre/ERT2 fusion gene, an frt-flanked kanamycin cassette were inserted directly at the ATG of the human CRH locus. These transgene is targeted as a single copy "knock-in" into the upstream region of the Hprt mutant locus. Transient infection with FLP recombinase removes the kanamycin cassette.