Fig. 1

Introducing ADAM10-uncleavable GD mutations into N-cadherin/Cdh2 in the mouse genome, and testing the mutant distribution in cultured cells. a Genomic DNA map of the mouse Cdh2 gene locus, mutant design, and targeting strategy. The gene targeting vector was designed to introduce tandem missense mutations (⁶⁶⁵⁶AGG ATC for ⁷¹⁴RI → GGG GAT for GD) in the exon 13 of the mouse Cdh2 gene. The targeting vector was introduced into C57BL/6-derived ES cells by electroporation. Neomycin-resistant clones were screened for homologous recombination with genomic Southern blotting using two probes, and one such clone was injected into ICR blastocysts. The resulting chimera mice were intercrossed with C57BL/6 N mice. The floxed neomycin-resistant cassette was removed from the allele by intercrossing the F1 mice with a line of CAG-Cre driver mice of C57BL/6 strain. The mutant and wild type alleles were discriminated routinely by PCR genotyping, using a pair of primers (arrowheads). See “Methods” for details. b Representative immunofluorescence images of FLAG-tagged, WT and GD mutant N-cadherin expressed in CHO cells. Their distribution patterns on ER, Golgi apparatus, and plasma membrane (green), as well as colocalization with endogenous β-catenin (red), were comparable. Nuclear DNA is stained with DAPI (cyan)