Gene targeting at the Nov locus. A. Schematic diagram showing wild type and targeted Nov alleles, the targeting construct, and the positions of the 5' and 3' external probes used to confirm targeting by Southern blotting. The TkneopolyA cassette replaces exon 3 in the targeted allele while the HSV TK cassette allows negative selection with Gancyclovir. B. Southern analysis of DNA from wild type, heterozygous and homozygous embryos using 5' and 3' external probes to confirm targeting. Digestion with BamHI gave a 16 kb wild type band with the 5'external probe and 14 kb targeted allele with the 5' probe, while digestion with EcoRI gave a 14 kb wild type band and 8.5 kb targeted band with the 3' probe. C. Western blotting of whole cell lysates and conditioned media from Novdel3homozygous and wild type E13.5 PEFs, using an anti-NOV antibody (59.3). Although NOV protein is readily detectable in wild type PEF whole cell lysates, no NOV protein can be detected in Novdel3homozygous PEFs. Conditioned medium from wild type PEFs contains high levels of secreted NOV, whereas that from Novdel3-/- PEFs contains trace amounts of mutant NOV protein lacking the VWD domain.