Generating a conditional allele of <i>Edn2</i> directly in DBA/2J-derived ES cells.

<p>(<b>A</b>) A targeting construct was generated that included <i>loxP</i> sites flanking exon 2 (e2) of the <i>Edn2</i> gene. Exon 2 contains part of the key functional domain for Edn2, and removal of exon 2 induces a frameshift for protein translation (<b>B</b>) Correct targeting at the 5′ end of the Edn2 gene was assessed using Xba1 restriction digests and southern blotting. The presence of the Neomycin (NEO) selectable marker incorporates an additional <i>Xba</i>1 restriction enzyme site in the targeted allele, generating a 6.6 kb fragment (Flox) compared to the normal 8.7 kb fragment (WT). Black arrows indicate clones correctly targeted at the 5′ end. (<b>C</b>) An example of a chimeric mouse generated as a result of injection of targeted DBA/2J ES cell clones into C57BL/6J blastocysts. (<b>D–E</b>) Chimeric male mice were mated with DBA/2J females and offspring genotyped for the heterozygous presence of the loxP sites in intron 1 (WT = 481 bp, Flox = 631 bp, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050081#s2" target="_blank">Methods</a>). White arrows indicate mice that carry the floxed allele of <i>Edn2</i>. (D). See methods for primer information. As expected, 100% of heterozygous mutant offspring had the DBA/2J coat color (dilute brown agouti, E).