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Fig. 1. Generation of PKN1 transgenic mice. (A) Structure of the construct used to generate transgenic mice. Binding sites for the primers Flag.for and PKN.rev used to identify transgenic animals are indicated. (B) Transgene expression in animals of line 933 and 995. RNA was isolated from mammary glands of lactating animals of the indicated lines and subjected to RT-PCR using the primers Flag.for and PKN.rev (PKN-KD). Bands for the murine β-actin transcript are shown as control (lower panel). (C) Expression of the Flag-tagged PKN1 protein in mammary glands of lines 933 and 995. Protein extracts from wild-type (WT) and transgenic animals of the indicated lines were immunoprecipitated with an antibody against the FLAG epitope tag and western blotting was performed with an antibody against the C-terminus of human PKN1. (D) Temporal expression pattern of the transgene in line 933. RNA was prepared from virgin animals (virg) and on pregnancy day ten (P10); lactation day 2 (L2); lactation, day ten (L10); involution, day five (I5); involution day ten (I10), involution day 60 (I60). Northern blot hybridization was conducted using a transgene specific probe covering the C-terminus of the human PKN1. The blot was subsequently hybridized with a probe for the S26 antigen as a loading control. Note that, compared with lactation, more RNA was necessary to obtain a hybridization signal in virgin and pregnant animals.
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