A targeting vector containing a Neo cassette and herpes simplex virus thymidine kinase gene was used to disrupt an exon which encodes the first C2 domain. The construct was electroporated into 129X1/SvJ derived RW-4 embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts. The resulting chimeric male animals were crossed to C57BL/6 female mice. The mice were backcrossed to C57BL/6 for several generations, then backcrossed to 129S6/SvEvTac for 7 or 8 generations (see SNP note below). Upon arrival at The Jackson Laboratory, mutant females and mutant males were bred together to establish the colony.
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. Markers on Chromosomes 8 and 13 are segregating, suggesting an incomplete backcross.
