The Pld1- null allele was created by Dr. Gilbert Di Paolo (Columbia University Medical Center). A targeting vector was designed to have a loxP site upstream of exon 13, and a frt-flanked neo cassette with 3' loxP site just downstream of exon 14 of the phospholipase D1 gene (Pld1) on chromosome 3. The construct was electroporated into C57BL/6 x 129/SvJ embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts, and chimeric males were bred with C57BL/6J females to establish the Pld1Flox Neo colony. The mice were then bred to the C57BL/6J-congenic FLP deleter strain (Stock No. 005703) for germline removal of the frt-flanked neo. The resulting Pld1Flox colony was backcrossed to C57BL/6J wildtype mice for several generations (and the Flp-expressing transgene was removed). The donating investigator reports that mice were bred with the C57BL/6-congenic Cre deleter strain (Stock No. 003724) for germline deletion of the floxed sequences (exons 13-14 and single frt site); creating the Pld1- null allele. The donating investigator reported the Pld1- colony had been backcrossed to C57BL/6J a total of eight generations (and the cre-expressing transgene was removed) prior to sending males with black coat color to The Jackson Laboratory Repository in 2016 (see SNP results 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).
Of note, the donating investigator reports that, at least once during backcrossing, a heterozygous female was bred to a C57BL/6J inbred male (thus the Y chromosome of the congenic strain should be of C57BL/6J origin).
In 2016, 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 mice sent to The Jackson Laboratory Repository, as well as a cohort of our first generation rederived mice. In both assays, this showed 2 of 27 markers were not fixed for C57BL/6 allele-type (i.e., still segregating for 129 allele-type markers): one on chromosome 3 (~10 Mbp away from Pld1 locus) and one on chromosome 13. In addition, 1 of 5 markers that determine C57BL/6J from C57BL/6N was found to be segregating (chromosome 13) - note the allele-type for C57BL/6N and 129S are the same for these 5 markers. Collectively, these data suggest that the mice sent to The Jackson Laboratory Repository were mostly C57BL/6J, and they retained a region of chromosome 13 that is derived from non-C57BL/6 origin.
