Figure 1.

Generation of MMTV-PTK6 transgenic mice. (a) A schematic diagram of the MMTV-PTK6 construct is shown. A 2.2 kb human PTK6 complementary DNA (gray region) was inserted into the third exon of the rabbit β-globin gene under the control of the MMTV LTR (striped region). (b) Expression of the MMTV-PTK6 construct transfected into NMuMG cells stimulated with dexamethasone. Transgenic PTK6 protein levels (NMuMG +Tg) are comparable to that produced in human breast cancer cell lines MCF7, MDA-MB-231 and MDA-MB-453. PTK6 was not detected in the MDA-MB-435 cell line. Expression of β-actin was examined as a loading control. (c) Ribonuclease protection assays were performed with RNAs prepared from mammary glands of three transgenic lines (B28, B33 and B35) and nontransgenic control mice (NT). PTK6 mRNA was detectable in the three transgenic lines but not in NT animals. Mouse cyclophilin was used as loading control. (d) Ectopic PTK6 expression was detected in transgenic mammary glands by immunoblotting. Levels of ectopic PTK6 protein expression correlated with the levels of PTK6 mRNA shown in c. (e). Immunohistochemistry demonstrates expression of ectopic human PTK6 in the transgenic mammary gland epithelial cells, as shown in the virgin animals (B28, B33 and B35) and pregnant animals (B33.Pg). The nontransgenic mammary gland stained negative for PTK6. Size bar=20 μm.