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Fig. 1. Generation of Ncad k.i. mice. (A) Targeting strategy with
schematic representation of the 5' region of the Ecad genomic
locus including promoter (P) and exons 1 and 2 (E1 and E2). Targeting vectors
and expected recombined alleles are shown. Sites for restriction endonucleases
and probes used are indicated. The neomycin selection cassette is flanked by
two loxP sites (red triangles). (B) Southern blot analysis of
targeted ES cell clones. DNA was digested with XbaI and probed with a
5' probe (upper panel) located upstream of the targeting vectors (see
also Fig. 1A). Fragments of 6.6 kb and either 7.8 kb or 8.5 kb were detected,
corresponding to wt and Ncad k.i. or Ncad-GFP k.i. alleles,
respectively. The internal probe was located downstream of exon 2. Fragments
of 8.8 kb and either 9.3 kb or 10.1 kb, corresponding to wt and Ncad
k.i. or Ncad-GFP k.i. alleles, respectively, were detected with
SpeI-digested DNA (lower panel). Corresponding genotypes are
indicated. (C) Double immunofluorescence against E- and N-cad on wt and
targeted ES cell clones. N-cad is expressed from the k.i. allele and
co-localizes with E-cad at the cell membrane. Wild-type ES cells are negative
for N-cad immunoreactivity. (D) RT-PCR analysis of Ncad-GFP
k.i. expression in different organs at E15.5. cDNAs from indicated organs of
wt (+/+) or Ecad+/Ncad (+/k.i.) embryos were used with
specific primers to amplify transcripts of Ecad, endogenous
Ncad and Ncad-GFP. (E) Immunohistochemistry of wt and
heterozygous Ncad k.i. embryos at E15.5. E-cad shows membrane
localization in the intestine epithelium of wt and heterozygous Ncad
k.i. embryos. N-cad k.i. protein is specifically detected in the intestinal
epithelium of embryos carrying one Ncad k.i. allele but not in wt
embryos (arrows). Endogenous N-cad protein is detected in peripheral muscle
cells of the intestine in both wt and heterozygous embryos (arrowheads). Scale
bar: 50 µm.
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