A targeting vector containing a FRT site flanked Neo selection cassette and a 3’ end loxP was utilized in the construction of this mutant. This selection cassette was inserted upstream of exon 76 of the targeted gene, and another loxP site was inserted downstream of exon 77. The construct was electroporated into 129P2/OlaHsd derived E14K embryonic stem (ES) cells.
Correctly targeted ES cells were injected into C57BL/6 blastocysts. The resulting chimeric animals were tested for germline transmission.
Heterozygotes were crossed with Flpe-deleter mice on the B6 genetic background to delete the FRT site flanked NEO cassette. Mice that retained the loxP site flanked exons 76 and 77 were then bred to C57BL/6 mice to remove the FLP, and were backcrossed to C57BL/6 for 6 generations. During backcrossing, the Y chromosome may not have been fixed to the C57BL/6 genetic background.
Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).
A 48 SNP (single nucleotide polymorphism) panel analysis, with 43 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 at The Jackson Laboratory Repository. While the 43 markers throughout the genome suggested a C57BL/6 genetic background, all 5 markers that determine C57BL/6J from C57BL/6N were found to be segregating. These data suggest the mice sent to The Jackson Laboratory Repository were on a C57BL/6N genetic background.
