Fig. 1. Development of NcadC transgenic mice. (A) Linearized structure of the OG2-NcadC construct used for transgenic expression. The 1.1 kb PstI fragment was used as a probe in Southern analysis, and the two arrows identify the primers used for PCR genotyping. Both the cDNA probe and the 250 kb PCR product encompass the junction between the OG2 promoter and the 5' end of the NcadC reading frame, thus making them specific for the transgene. (B) Southern blot (upper panel) and PCR (lower panel) of genomic DNA of one mouse litter showing a 1.1 kb band or a 250 bp product, respectively, corresponding to the transgene. (C) Detection of transgene expression by reverse transcription and PCR of total RNA extracted from tissues of 2-month-old transgenic animals, showing a 195 bp band corresponding to NcadC mRNA only in extracts from calvaria and femurs, but not from other tissues. Control reactions were run either in the absence of reverse transcriptase (No RT) or in the presence of NcadC plasmid, as indicated. Amplification of GAPDH was used to control for mRNA integrity and PCR efficiency. (D) Total protein extracts of mouse calvaria isolated from either OG2-NcadC (Tg) or wild-type (WT) animals were immunoprecipitated (IP) using the PEP.1 antibody that recognizes the intracellular tail of Xenopus N-cadherin. Proteins in the whole-cell lysate (input) and in the immunoprecipitate (beads) were separated by SDS-PAGE and blotted (WB) using an anti-FLAG antibody. A band of 45 kDa, corresponding to the truncated NcadC, was detected in the immunoprecipitate from transgenic animals, but not from wild-type littermates. The higher molecular weight bands represent IgG heavy chain.