A targeting vector was designed to insert loxP sites flanking exon 2, which contains the entire coding region of the basic helix-loop-helix family, member a15 (Bhlha15) gene. The MIST1 coding region was also engineered to contain an E. coli biotin ligase BirA biotin tag (BT) sequence (MASSLRQILDSQKMEWRSNAGGS) at the N-terminus of the protein where BirA can biotinylate the central lysine residue in the sequence. Additionally, the MIST1 coding region contains a MYC/6x His tag at the C-terminus that can be detected using anti-MYC. A frt-flanked neomycin resistance (neo) cassette was inserted downstream of the floxed sequence. Of note, the floxed region has been sequenced and appears to be derived from rat. The construct was electroporated into (129X1/SvJ x 129S1/Sv)F1-Kitl+-derived R1 embryonic stem (ES) cells. Correctly targeted ES cells were injected into blastocysts and resulting chimeric males were bred with C57BL/6NHsd females. The donating lab reported that the resulting offspring were bred to C57BL/6NHsd mice for at least 20 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/6NJ oocytes (Stock No. 005304).
Of note, this strain arrived carrying the C57BL/6NHsd linked Dock2m1Hsd spontaneous mutation. This allele has since been removed from the colony maintained at The Jackson Laboratory.
A 48 SNP (single nucleotide polymorphism) panel analysis, 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, was performed on the rederived living colony at The Jackson Laboratory Repository. Two of the 27 markers throughout the genome were segregating with 129, suggesting an incomplete backcross. Also, 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.
