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).
Elizabeth M Simpson, Centre for Molecular Medicine & Therapeutics, University of British Columbia
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/ERT2 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/ERT2 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.
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 Hprtb-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-Aw-j/Aw-j Hprtb-m3/Y mice (created by four generations of breeding C57BL/6NTac-Aw-J together with C57BL/6NTac-Hprtb-m3). The parental lines were individually made by backcrossing C57BL/6J-Aw-J/J (Stock No. 000051) four generations onto C57BL/6NTac, and backcrossing B6.129P2-Hprtb-m3/J (Stock No. 002171) six generations onto C57BL/6NTac, respectively.
|Expressed Gene||cre/ERT2, Cre recombinase and estrogen receptor 1 (human) fusion gene,|
|Site of Expression|
|Allele Name||targeted mutation 353, Elizabeth M Simpson|
|Allele Type||Targeted (Recombinase-expressing, Inducible)|
|Allele Synonym(s)||CRH-creERT2; mEMS5758; Ple274-icre/ERT2|
|Gene Symbol and Name||Hprt, hypoxanthine guanine phosphoribosyl transferase|
|Expressed Gene||cre/ERT2, Cre recombinase and estrogen receptor 1 (human) fusion gene,|
|Strain of Origin||B6N.Cg-Aw-J Hprtb-m3|
|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 |
The targeted mutation in on the X chromosome. When maintaining a live colony, heterozygous females may be bred with wildtype males from the colony or with C57BL/6J inbred males (Stock No. 000664). Alternatively, wildtype females from the colony or C57BL/6J inbred females may be bred with hemizygous males. Homozygous females and hemizygous males are expected to be viable and fertile. The expected coat color is black.
When using the B6.Cg-Hprttm353(Ple274-icre/ERT2)Ems/Mmjax mouse strain in a publication, please cite the originating article(s) and include MMRRC stock #037376 in your Materials and Methods section.
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