The Lang-EGFP targeting vector was designed to contain an internal ribosome entry site (IRES), and an enhanced green fluorescent protein (EGFP), followed by a self-excising loxP-flanked ACN cassette (itself containing a neomycin resistance (Neo) gene and a testes-specific angiotensin-converting enzyme promoter-driven Cre recombinase gene), which self-excises during transmission in the male chimera. This construct was inserted downstream of the internal stop codon of the CD207 antigen (Cd207 or Langerin) gene. This construct was injected into 129S2/SvPas-derived CK35 embryonic stem (ES) cells. Correctly targeted ES cells were injected into BALB/c blastocysts, and the resulting chimeric males were crossed to C57BL/6 females. The donating investigator reported the mice were backcrossed to C57BL/6 mice for at least 15 generations (see SNP results below) prior to sending males to The Jackson Laboratory Repository in 2012. Upon arrival, males were used to cryopreserve sperm. To establish the living mouse colony, an aliquot of the frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).
In 2013, 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, all 5 markers that determine C57BL/6J from C57BL/6N were found to be segregating. These data show that the mice sent to The Jackson Laboratory Repository were on a C57BL/6N genetic background, and the living colony in 2103 is on a mixed C57BL/6N;C57BL/6J genetic background.
