A targeting vector designed with (from 5' to 3') a splice acceptor site, a loxP-flanked Neo-STOP cassette, a cDNA sequence encoding rat MEK1DD (a mutant form of MAPKK1 rendered constitutively active by two serine->aspartic acid substitutions (S218D/S222D) within the catalytic domain], an frt-flanked internal ribosomal entry site-enhanced green fluorescent protein cassette (IRES-EGFP), and bovine polyadenylation signal. This transgene construct was inserted between exons 1 and 2 of the Gt(ROSA)26Sor locus via electroporation into C57BL/6-derived embryonic stem (ES) cells. Correctly targeted ES cells were selected and chimeric animals were bred to C57BL/6 to generate the mutant colony. The donating investigator reports that these R26StopFLMEK1DD mice were maintained on a C57BL/6 genetic background (see SNP note below) as a homozygous colony for many generations prior to arrival at The Jackson Laboratory Repository. Upon arrival, mutant mice were bred to C57BL/6J inbred mice (Stock No. 000664) for at least one generation to establish the colony.
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. In some of the mice, one marker on chromosome 11 (~8.4 Mbp) and one marker on chromosome 17 (~34.3 Mbp) were not fixed to the C57BL/6 genetic background. The source of this is not known. In addition, at least 3 of 5 markers that determine C57BL/6J from C57BL/6N were found to be segregating in all the rederived mice. These data suggest the mice sent to The Jackson Laboratory Repository were on a mixed C57BL/6J ; C57BL/6N genetic background.
