Pi4ka^tm2.1Arte

A targeting vector was designed to insert a loxP site upstream of exon 48 of the phosphatidylinositol 4-kinase, catalytic, alpha polypeptide (Pi4ka) gene. A human growth hormone polyadenylation signal (hGHpA) and a second loxP site followed by a frt/F3-flanked neomycin (neo) resistance cassette were placed downstream of exon 55 of Pi4ka. Using a recombination-mediated exchange cassette (RMCE), correctly targeted ES cells were then electroporated with a second targeting vector. This second targeting vector replaces the frt/F3-flanked neo cassette with a attB/attP-flanked puromycin (puro) resistance cassette and loxP site followed by a duplicated genomic region containing exons 48 to 55. The puro cassette and the duplicated region were flanked by frt/F3 sites. The duplicated genomic region contained a point mutation in exon 51 resulting in an Arginine to Lysine substitution (R1900K). PhiC31-mediated recombination removed the puro cassette. (J:172498)