To generate the Adipoq-Cre BAC transgenic mice, the 245 kb RP23-90G21 bacterial artificial chromosome (BAC) containing at least five mouse genes was modified by replacing the starting ATG and 222 bp of the adiponectin gene with the starting ATG and coding sequence of a Cre recombinase gene. This modified BAC transgene was microinjected into pronuclei of fertilized one-cell stage FVB/NJ embryos. Transgenic mice were identified and bred to C57BL/6J wildtype mice to establish founder lines. A single transgenic founder line was characterized. The donating investigator reported that transgenic mice were then backcrossed to C57BL/6J wildtype mice for more than 14 generations prior to sending to The Jackson Laboratory Repository (see 2011 SNP notes below). Upon arrival, mice were bred to C57BL/6J inbred mice (Stock No. 000664) to establish our live colony. By 2014, the colony was additionally backcrossed to C57BL/6J inbred mice for at least five generations and confirmed by SNP analysis (below) to be C57BL/6J congenic.
In 2011, a 32 SNP (single nucleotide polymorphism) panel analysis, with markers covering all 19 chromosomes and the X chromosome, was performed on the rederived living colony at The Jackson Laboratory Repository. This revealed six markers representing six different chromosomes that were not fixed as C57BL/6 allele-type. Five of these markers, one each on chromosomes 2, 8, 9, 15, and 19, are segregating for C57BL/6 or FVB allele-type. The marker at ~88.2 Mbp on chromosome 16 (that is not polymorphic between C57BL/6 and FVB) was found to be heterozygous for C57BL/6 and an undefined allele-type (i.e., not FVB) in all first generation rederived mice tested. Collectively, these data suggest the mice may not have been backcrossed to C57BL/6 for as many generations as originally reported prior to arrival at The Jackson Laboratory Repository. In addition, the chromosome 16 marker represents an unknown source of genetic contamination.
By 2014, the same 32 SNP panel assay was performed on a cohort of hemizygous females (from several litters) from our colony after five generations of backcrossing onto C57BL/6J. This revealed all markers but one were C57BL/6 allele-type. The single heterozygous (C57BL/6J;FVB/NJ) marker is on chromosome 9 (~53 cM). These data suggest the colony is now C57BL/6J congenic, and the transgene insertion site(s) may be on chromosome 9.
In Wong et al. 2021 Adipocyte 10:21, hemizygous Adipoq-Cre mice obtained from The Jackson Laboratory Stock No. 028020 were found to have the Adipoq-Cre transgene integrated on chromosome 9 between exons 6 and 7 of the Tbx18 locus, an embryonic development gene also associated with myocardial function. No genomic rearrangements were detected in the region of the integration site, although the insertion leads to reduced Tbx18 expression in hemizygous epididymal white adipose tissue. While the targeted locus amplification mapping predicted transgene-transgene fusions had occurred between different parts of the transgene, their ddPCR analysis identified the number of copies of intact Cre recombinase as one per genome. This publication also reported that when breeding hemizygous mice together, they were unable to generate any offspring with 2 copies of Cre recombinase (possible lethality of homozygotes).
