(The) construction of targeting vector for the disruption of the PLC beta 3 gene

Abstract

Phosphoinositide-specific phospholipase C(PLC) is a crucial enzyme in transmembrane signaling. Six mammalian PLC isozymes including PLC-beta 1, PLC-beta 2, PLC-gamma 1, PLC-gamma 2, PLC-delta 1, and PLC-delta 2 have been identified at both protein and DNA levels. Until now, although the biochemical activities of the PLC isotypes have been known, the physiological role of the PLCs is not elucidated. Among these PLC isotypes, to understand the physiological role of PLC beta 3 enzyme, mouse null mutant that has the disrupted PLC-beta 3 gene was designed. As a first step, using rat PLC beta-3 cDNA as a probe, about 10kb lambda clones were obtained from the mouse Balb/C genomic library in EMBL3, and then 5.4kb PLC beta 3 genomic DNA was subcloned into pUC19. Through the restriction enzyme digestion and sequence analysis, sequence information and restriction map of the PLC beta 3 gene were elucidated. Targeting vector for the disruption of the PLC beta 3 gene was constructed. It was made of three components; negative selection marker : thymidine kinase gene, positive selection marker : neo-resistant gene, homologous integration site : PLC beta 3 genomic DNA fragment(5kb) without exon 5 - containing region(0.4kb). Total length of the targeting vector was about 12kb. Through the electroporation of the targeting vector into TT2 embryonic stem(ES) cells, recombinant ES cells were obtained. We are looking for the homologous recombinant clones using PCR and Southern blotting.