Generation of EcNc and NcEc cadherin proteins expressed in the <i>E-cad</i> (<i>Cdh1</i>) locus.

<p>(A) Schematic representation of EcNc and NcEc protein structure in their cadherin-catenin complex (adapted from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002609#pgen.1002609-Stemmler1" target="_blank">[8]</a>). (B) Gene targeting strategy and the resultant knock-in allele, representatively shown for NcEc; TK, <i>HSV</i>::<i>tk</i> negative selection cassette; HA, haemagglutinin tag; pA, SV40 polyadenylation signal; Neo, neomycin resistance cassette, flanked by loxP sites (black triangles). (C) Southern blot analysis of obtained ES cell clones using the 5′ probe. (D) Expression of the knock-in alleles in ES cells after removal of the neomycin cassette reveals equal expression of both HA-tagged proteins. (E) Immunofluorescence labeling of EcNc (upper) and NcEc (lower panel) showing complete overlap of anti-HA and anti-E-cad staining in heterozygous E3.5 blastocysts in confocal optical sections. Scale bar, 25 µm.