A targeting vector was designed by Dr. Omar Abdel-Wahab (Memorial Sloan Kettering Cancer Center) to insert a loxP site upstream of exon 1 of the serine/arginine-rich splicing factor 2 (Srsf2) gene. cDNA encoding the sequence of exon 3, and an SV40 polyadenylation sequence bound to a frt-flanked neomycin resistance (neo) cassette, followed by a second loxP site were inserted downstream of exon 2. Duplicate exons 1-2 were inserted downstream of the neo cassette. This second exon 1 contains a point mutation in amino acid 95 which alters the nucleotide sequence from CCG to CAC, resulting in an amino acid change from Proline to Histidine. The cDNA for exon 3 was fused in frame to endogenous exon 2. The construct was electroporated into C57BL/6NTac-derived IC1 embryonic stem (ES) cells. Correctly targeted ES cells were injected into Balb/c blastocysts and resulting chimeric mice were bred with B6.Cg-Tg(ACTFLPe)9205Dym/J transgenic mice (Stock No. 005703) to delete the neo cassette. Resulting progeny were crossed to remove the Flp-expressing transgene, and Srsf2P95H floxed offspring were backcrossed to C57BL/6 mice for at least 6 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, 4 of 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 mixed C57BL/6J ; C57BL/6N genetic background.
