A targeting vector was used to insert a FRT site flanked PGK-NEO cassette and an IRES- CreERT2 into the 3' UTR of the Sftpc gene. The construct was electroporated into unspecified 129S6/SvEvTac derived 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 bred to FLP recombinase expressing mice (Stock No. 007844), on the 129S4/SvJae background, to remove the PGK-NEO selection cassette. The mice were then backcrossed to C57BL/6NCr (National Cancer Institute) for several generations. Next, the mice were sent to Dr. Jason Rock (University of California, San Francisco), where they were reported to have been backcrossed onto C57BL/6J for more than eight generations (see SNP results below). In 2016, Dr. Rock sent one Sftpc-CreERT2 male to The Jackson Laboratory. Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6NJ oocytes (Stock No. 005304).
In 2016, a 35 SNP (single nucleotide polymorphism) panel analysis, with 30 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 a cohort of first generation rederived living colony at The Jackson Laboratory Repository. The 30 markers throughout the genome all show C57BL/6 genetic background. For the 5 markers that determine C57BL/6J from C57BL/6N, half of the mice were homozygous C57BL/6N for all 5 marker, while the other half were homozygous C57BL/6N for 4 markers and heterozygous B6N;B6J for the other marker. These data suggest that the mice sent to The Jackson Laboratory Repository were on a mixed C57BL/6N;C57BL/6J genetic background (predominantly C57BL/6N).
Subsequent SNP testing was performed yearly from 2017-2020 on cohorts from the live colony using a 48 SNP panel, 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. The 43 markers throughout the genome all show C57BL/6 genetic background, and the C57BL/6 substrain markers all show homozygous C57BL/6N.
