Figure 1.
Targeted disruption of the mouse P2X4 gene. A, Maps of the P2X4 wild-type allele, targeting vector, and mutant allele. Boxes represent open reading frames; LacZ gene insertion upstream of the P2X4-initiating methionine resulted in the deletion of all the coding region of exon 1 and of the first exon–intron splice site. TK, Thymidine kinase; NEO, neomycin; Ex1, exon 1; B, BamH1; Bg, BgIII; S, SacI; X, XbaI. Southern blotting probe was designed outside of the targeting locus between 5′ SacI and XbaI sites. As shown, after digestion with SacI, this probe will hybridize to a 5 or 10 kb DNA fragment for wild-type and mutant allele, respectively. B, Southern blot analysis of genomic DNA digested with SacI from +/+, +/−, and −/− mice using the probe described in A. C, Western blot of membrane protein fraction from brain, lung, and submandibular gland of wild-type P2X4+/+ mice showed a band at 60 kDa, similar to that observed in P2X4-expressing HEK293 cells. No band of equivalent size was observed from knock-out P2X4−/− mice.