The P110*-transgene is a targeting vector designed with (from 5' to 3') a splice acceptor site, a loxP-flanked Neo-STOP cassette,
a cDNA sequence encoding the constitutively active P110*, an frt-flanked internal ribosomal entry site-enhanced green fluorescent protein cassette (IRES-EGFP), and bovine polyadenylation signal. P110* is a mutant form of mouse P110α (Pik3ca; the catalytic subunit of phosphatidylinositol 3-kinase) that was made constitutively active by addition of the p85 iSH2 domain (region between the two Src homology 2 domains of p85 that is required for the enzymatic activity of p110) fused to the p110 N terminus using a flexible glycine-kinker hinge region. 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. These R26StopFLP110* 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. 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.
