B6.Cg-Hprt^{tm352a(Hspa1a-icre/ERT2)Ems}/Mmjax

Stock number: 023697
Product name: Hspa1a-icre/frt/ERT2/frt;mEMS6051
Strain synonyms: Ple291-icre/frt/ERT2/frt;mEMS6051, C57BL/6-Hprt^{tm352(Ple291-icre/ERT2)Ems}/Mmjax, Hspa1a-creERT2

Description

Hspa1a-icre/frt/ERT2/frt;mEMS6051 mice have the Hspa1a-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 mouse heat shock protein 1A (Hspa1a) 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.

Detailed description

Hspa1a-icre/frt/ERT2/frt;mEMS6051 mice have the Hspa1a-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 mouse heat shock protein 1A (Hspa1a) 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 (Telencephalon, Hippocampus, Diencephalon)." 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. The iCre/ER^T2 fusion protein used here has also been modified to have F3-frt sites flanking the estrogen receptor sequences.

In 2014, a 32 SNP (single nucleotide polymorphism) panel analysis, with 27 markers covering all 19 chromosomes and the X chromosome, as well as 5 markers that distinguish between the C57BL/6J and C57BL/6N substrains, was performed on the first generation rederived living colony at The Jackson Laboratory Repository. While the 27 markers throughout the genome suggested a C57BL/6 genetic background, 4 of 5 markers that determine C57BL/6J from C57BL/6N were found to be segregating in some mice (chromosomes 8 [~15.2 Mbp], 11 [~4.4 Mbp], 13 [~41.0 Mbp] and 15 [~57 Mbp]). These data suggest that the mice sent to The Jackson Laboratory Repository were on a mixed C57BL/6J;C57BL/6N genetic background.

Development

Hspa1a-icre/frt/ERT2/frt;mEMS6051 mice were created by the CanEuCre and Pleiades Promoter Project (Centre for Molecular Medicine and Therapeutics, University of British Columbia). These mice have the Hspa1a-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 mouse heat shock protein 1A (Hspa1a) 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 below.

The Hspa1a-icre/frt/ERT2/frt transgene (pEMS2012) was designed with an HS4 insulator region, the Hspa1a promoter (878 bp from a 5' flanking region from the mouse heat shock protein 1A locus (Hspa1a or hsp68) known to contain five HSE and to be sufficient for proper heat induction of the hsp68 gene), a chimeric intron cassette, the iCre/ER^T2 fusion gene (with F3-frt sites flanking the ER^T2 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 Cre recombinase variant (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 ER^T2 (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 Hspa1a-icre/frt/ERT2/frt transgene was targeted as a single copy knock-in to the Hprt^b-m3 mutant locus on the X Chromosome via electroporation into mEMS4855.02 embryonic stem cells (described below). Chimeric males (founder line mEMS6051) were bred to C57BL/6J females to establish the mutant colony. The donating investigator reports that Hspa1a-icre/frt/ERT2/frt;mEMS6051 mice were backcrossed to C57BL/6J for 2 generations (see SNP results in strain description texts), and then sperm was frozen from black hemizygous males at UBC. 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 C57BL/6J oocytes (Stock No. 000664); the resulting obligate heterozygous females are bred at least one generation to C57BL/6J mice.

mEMS4855.02 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: Hprt^{tm352a(Hspa1a-icre/ERT2)Ems}
Name: targeted mutation 352a, Elizabeth M Simpson
MGI accession ID: MGI:5532520
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 was designed with an HS4 insulator region, the 878 bp Ple291 minipromoter (878 bp from a 5 prime flanking region from the mouse heat shock protein 1A locus (Hspa1a or hsp68) known to contain five HSE and to be sufficient for proper heat induction of the hsp68 gene), 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.