A targeting vector containing a FRT site flanked Neo selection cassette was utilized in the construction of this mutant. This selection cassette and a loxP site was inserted downstream of the first protein coding exon of the targeted gene, and another loxP site was inserted upstream of the first protein coding exon. This construct was electroporated into (129X1/SvJ x 129S1/Sv)F1-Kitl+ derived R1 embryonic stem (ES) cells. Correctly targeted ES cells were injected into C57BL/6 blastocysts. The resulting netrin-1flox/+ chimeric animals were crossed to mice expressing FLP1 recombinase under the control of the human beta actin promoter (Stock No. 003800). Mice that no longer carried the NEO cassette and retained the loxP site flanked first protein coding exon were then bred to C57BL/6 mice for 7 generations to remove the Tg(ACTFLPe)9205Dym transgene. The mice were then bred to germline Cre deleter transgenic mice (Stock No. 006054) to excise the first protein coding exon.
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, all 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 C57BL/6N genetic background.
