Analysis of cortactin transgene expression. (A) Structure of the MMTV-cortactin transgene construct. Vertical striped bar, 510 bp promoter/enhancer of the MMTV-LTR (-360/+150); stippled bar, 920 bp β-globin intron 2 sequence inserted to generate a stable transgene mRNA; black bar, 1.8 kb human EMS1 cDNA ; hatched bar, transcriptional processing sequences derived from SV40. Relevant restriction sites are indicated. (B) Southern blot analysis of tail DNA of three cortactin transgenic founders and one wild type (WT) mouse. The intensity of the bands corresponds to the increased copy number of the integrated transgenic cortactin cDNA. T6 contains one cDNA copy. (C) Northern blot analysis of 10-days lactating mammary glands. Expression of human transgenic cortactin mRNA (3,6 kb) was detected in T16 only. Full-length human cortactin cDNA was used as a probe. (D) Western blot analysis of 10-days lactating mammary glands to detect transgenic cortactin protein expression. Polyclonal antibody RA444 was used to recognize specific human transgene expression, whereas polyclonal antibody RA23 recognized both human and endogenous mouse cortactin. (E) Virgin T16 transgenic (T) and non-transgenic (W) female mice were treated with glucocorticosteroid dexamethasone to stimulate transgene expression from the MMTV promoter. Lysates from liver (Li), salivary gland (SG), kidney (Ki), spleen (Sp), pancreas (Pa), lung (Lu) and 10-days lactating mammary gland as a positive control (MG) were subjected to Western blotting. Polyclonal antibody RA444 was used to detect specific human transgene expression, whereas monoclonal 4F11 antibody recognizes both human and mouse cortactin. Transgene expression was significantly induced in the pancreas, liver and kidney.