A targeting vector was designed to insert a loxP upstream of exon 4 (containing the start codon), and a frt-flanked neomycin resistance (neo) cassette, followed by a second loxP site, downstream of exon 5 of the small glutamine rich tetratricopeptide repeat containing alpha (Sgta) gene. The construct was electroporated into C57BL/6-derived embryonic stem (ES) cells. Correctly targeted ES cells were injected into blastocysts and resulting chimeric males were bred with C57BL/6 females. Resulting Sgtawt/flox offspring were bred to OzCre mice, expressing Cre Recombinase from the Gt(ROSA)26Sor locus on a C57BL/6 background, to remove the floxed sequence and the neo cassette. Resulting mice were crossed to C57BL/6 mice to remove the cre gene, and Sgta+/- mice were backcrossed to C57BL/6 mice for at least 12 generations (see SNP note below). 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 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 at The Jackson Laboratory Repository. While the 27 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.
