A targeting vector was designed by Dr. Yinon Ben-Neriah (The Hebrew University of Jerusalem) to place loxP sites flanking exons 1-2 of the casein kinase 1, alpha 1 gene (Csnk1a1 or CKIα) on chromosome 18. This also inserted a loxP-flanked neomycin cassette. The construct was electroporated into (129X1/SvJ x 129S1/Sv)F1-Kitl+-derived R1 embryonic stem (ES) cells. Next, ES cells were transiently transfected with the pCA-NLS-Cre vector. The resulting ES cells with the CsnK1a1fl genotype (retaining the floxed exons 1-2 with deletion of the neo cassette) were identified and used for aggregation with CD1-derived morulae. Chimeric mice were bred to CD1 for germline transmission. The donating investigator reported that the CsnK1a1fl colony was bred with C57BL/6 inbred mice (a mixture of C57BL/6JOlaHsd and C57BL/6J) for at least ten generations (see SNP results below). During backcrossing, the Y chromosome may not have been fixed to the C57BL/6 genetic background. In 2015, CsnK1a1fl males were sent to The Jackson Laboratory Repository. Upon arrival, sperm was cryopreserved. To generate the living CsnK1a1fl colony, an aliquot of the frozen sperm was used to fertilize oocytes from C57BL/6J female mice (Stock No. 000664). The CsnK1a1fl colony was then maintained by breeding heterozygous or homozygous mice together.
In 2015, 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 all 14 heterozygous mice from the first generation rederived living colony at The Jackson Laboratory Repository. All 26 markers on somatic chromosomes throughout the genome suggest a C57BL/6 genetic background. All heterozygous females, but none of the heterozygous males, had one marker on the X chromosome [~66.9 Mbp] segregating for C57BL/6 and 129 allele-type.
In addition, 3 of 5 markers that determine C57BL/6J from C57BL/6N were found to be segregating in all tested mice (chromosomes 8 [~15.2 Mbp], 15 [~57.6 Mbp] and 19 [~49.9 Mbp]), and some mice had one marker segregating on chromosome 13 [~41.0 Mbp]. These data suggest that the mice sent to The Jackson Laboratory Repository were on a mixed C57BL/6N;C57BL/6J genetic background, or alternatively, the C57BL/6JOlaHsd contributions may have similar SNPs as C57BL/6N.
