Sed 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 HI gave a 16 kb wild type band with the 5'external probe and 14 kb targeted allele with the 5' probe, while digestion with RI 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 homozygous 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 homozygous PEFs. Conditioned medium from wild type PEFs contains high levels of secreted NOV, whereas that from -/- PEFs contains trace amounts of mutant NOV protein lacking the VWD domain.<p><b>Copyright information:</b></p><p>Taken from "Abnormal skeletal and cardiac development, cardiomyopathy, muscle atrophy and cataracts in mice with a targeted disruption of the () gene"</p><p>http://www.biomedcentral.com/1471-213X/8/18</p><p>BMC Developmental Biology 2008;8():18-18.</p><p>Published online 20 Feb 2008</p><p>PMCID:PMC2275724.</p><p></p
