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.
