Ple155-icre/frt/ERT2/frt;mEMS5985 mice have the Ple155-icre/frt/ERT2/frt 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 specific portions from the promoter/enhancer/regulatory regions of the human purkinje cell protein 2 (PCP2) locus to direct expression of a tamoxifen-inducible, improved Cre recombinase (icre/ERT2). In these mice, the icre/ERT2 estrogen receptor sequences are flanked with frt sites.
Elizabeth M Simpson, Centre for Molecular Medicine & Therapeutics, University of British Columbia
Genetic Background | Generation |
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Allele Type | Gene Symbol | Gene Name |
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Targeted (Recombinase-expressing, Inducible) | Hprt | hypoxanthine guanine phosphoribosyl transferase |
Ple155-icre/frt/ERT2/frt;mEMS5985 mice have the Ple155-icre/frt/ERT2/frt 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 specific portions from the promoter/enhancer/regulatory regions of the human purkinje cell protein 2 (PCP2) locus directing expression of the tamoxifen-inducible, improved Cre recombinase (icre/ERT2). In these mice, the icre/ERT2 estrogen receptor sequences are flanked with frt sites. The donating investigator reports RT PCR expression as "Positive (Eyes, Diencephalon, Cerebellum)." 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. The iCre/ERT2 fusion protein used here has also been modified to have F3-frt sites flanking the estrogen receptor sequences.
Ple155-icre/frt/ERT2/frt;mEMS5985 mice were created by the CanEuCre and Pleiades Promoter Project (Centre for Molecular Medicine and Therapeutics, University of British Columbia). These mice have the Ple155-icre/frt/ERT2/frt 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, specific regions from the promoter/enhancer/regulatory sequences of the human purkinje cell protein 2 (PCP2) locus are designed to direct expression of the tamoxifen-inducible, improved Cre recombinase (icre/ERT2). In these mice, the icre/ERT2 estrogen receptor sequences are flanked with frt sites. More specific details are described below.
The Ple155-icre/frt/ERT2/frt transgene (pEMS2004) was designed with an HS4 insulator region, the 1652 bp Ple155 minipromoter (based on PCP2-B; derived from a portion of the endogenous promoter (Prom 1 and Prom 2) from the human purkinje cell protein 2 (PCP2) locus), a chimeric intron cassette, the iCre/ERT2 fusion gene (with F3-frt sites flanking the ERT2 sequences), the mut6-WPRE element, an SV40 early polyA signal, an additional HS4 insulator region, 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. The iCre/ERT2 fusion gene used here has an optimized variant of Cre recombinase (iCre; improved with mammalian codon usage, removed putative cryptic splice sites, altered stop codon, and reduced CpG content to limit the chances of epigenetic silencing in mammals) fused to ERT2 (a G400V/M543A/L544A triple mutation of the human estrogen receptor ligand binding domain); additionally modified to have F3-frt sites flanking the estrogen receptor sequences. The Ple155-icre/frt/ERT2/frt transgene was targeted as a single copy knock-in to the Hprtb-m3 mutant locus on the X Chromosome via electroporation into mEMS4855.02 embryonic stem cells (described below). The resulting chimeric males (founder line mEMS5985) were bred to C57BL/6J females to establish the mutant colony. The donating investigator reports that Ple155-icre/frt/ERT2/frt;mEMS5985 mutant animals were backcrossed to C57BL/6J mice for 2 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.
mEMS4855.02 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, |
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Site of Expression |
Allele Name | targeted mutation 347a, Elizabeth M Simpson |
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Allele Type | Targeted (Recombinase-expressing, Inducible) |
Allele Synonym(s) | PCP2-creERT2; Ple155-icre/frt/ERT2/frt;mEMS5985 |
Gene Symbol and Name | Hprt, hypoxanthine guanine phosphoribosyl transferase |
Gene Synonym(s) | |
Expressed Gene | cre/ERT2, Cre recombinase and estrogen receptor 1 (human) fusion gene, |
Strain of Origin | B6N.Cg-Aw-J Hprtb-m3 |
Chromosome | X |
Molecular Note | The transgene was designed with an HS4 insulator region, the 1652 bp Ple155 minipromoter (based on PCP2-B; derived from a portion of the endogenous promoter (Prom 1 and Prom 2) from the human purkinje cell protein 2 (PCP2) locus), a chimeric intron cassette, the iCre/ERT2 fusion gene (with F3-frt sites flanking the ERT2 sequences), the mut6-WPRE element, an SV40 early polyA signal, an additional HS4 insulator region, 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. T |
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-Hprttm347a(Ple155-icre/ERT2)Ems/Mmjax mouse strain in a publication, please cite the originating article(s) and include MMRRC stock #037077 in your Materials and Methods section.
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The Jackson Laboratory has rigorous genetic quality control and mutant gene genotyping programs to ensure the genetic background of JAX® Mice strains as well as the genotypes of strains with identified molecular mutations. JAX® Mice strains are only made available to researchers after meeting our standards. However, the phenotype of each strain may not be fully characterized and/or captured in the strain data sheets. Therefore, we cannot guarantee a strain's phenotype will meet all expectations. To ensure that JAX® Mice will meet the needs of individual research projects or when requesting a strain that is new to your research, we suggest ordering and performing tests on a small number of mice to determine suitability for your particular project. We do not guarantee breeding performance and therefore suggest that investigators order more than one breeding pair to avoid delays in their research.
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