A 3.0Kb region of the Crh promoter was PCR amplified from mouse bacterial artificial chromosome clone #129A14 and resulting amplicons were topo-cloned into pCR2.1-topo, (Invitrogen, Carlsbad, CA). Incorporated into the primers were restriction sites for later subcloning. The lentivirus backbone, pCMVGFPdNhe was digested with ClaI and SalI to remove the CMVGFP sequence, The linearized lentivirus backbone was ligated to the 3.0Kb Crh promoter (a ClaI/BamHI fragment) and to the Cre coding sequence, (a BamHI/SalI fragment). The LVCRFp3.0Cre construct was linearized with NheI and SphI, purified by electroelution, and diluted to 2ng/ul for pronuclear microinjection into FVB mouse cells by the Emory University Transgenic Core Facility. The donating investigator reported that this strain was backcrossed approximately 4-6 times to C57BL/6 (see SNP results below) prior to sending males to The Jackson Laboratory Repository in 2011. Upon arrival, males were used to cryopreserve sperm. To establish the living mouse colony, an aliquot of the frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).
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 rederived living colony (F2 generation) at The Jackson Laboratory Repository. This revealed 6 of 27 markers, one each on chromosomes 2, 6, 7 and 9, and two markers on chromosome 17, that were not fixed for C57BL/6 allele-type (e.g., still segregating for FVB/N allele-type markers). In addition, all 5 markers that determine C57BL/6J from C57BL/6N were also found to be segregating on chromosomes 8, 11, 13 and 19 (note that these markers are the same for C57BL/6N and FVB/N). These data suggest the mice were backcrossed to C57BL/6 for fewer generations than reported prior to arrival at The Jackson Laboratory Repository, and the mice are most likely now a mix of C57BL/6 (~65-78%) and FVB/N (~22-35%).
