Generation of the MTB/Cat^TMILA bigenic mouse.

(A) Design of the TetO- ΔN89β-Cat^TMILA transgene. The ΔN89β-catenin cDNA (2.1kb) was cloned into a single EcoR1 restriction site downstream of the TetO sequence in the TMILA (7.4kb) cloning vector [11]. The ΔN89β-catenin cDNA encodes the truncated Xenopus β-catenin protein with a myc-epitope tag fused in-frame at its N-terminus (black box). The location of the PCR primers for genotyping (black arrowheads) as well as the 13 centrally located Armadillo repeats (Arm repeats) is indicated. The inserted ΔN89β-catenin cDNA is followed by an IRES and a cDNA encoding the firefly luciferase protein. A SV40 polyadenylation signal (PA) serves as a strong transcriptional termination signal. The TetO- ΔN89β-Cat^TMILA transgene was linearized with Not1, isolated from vector sequences, and purified prior to pronuclear microinjection. (B) Schematic depicts the breeding strategy to generate the MTB/Cat^TMILA bigenic mice by crossing the MTB effector transgenic [14] with TetO-ΔN89β-Cat^TMILA responder transgenic. (C) Typical western immunoblot of isolated mammary epithelial cell protein. Lane 1, 2, and 3 denote mammary epithelial protein isolated from wild type ((WT) or non-transgenic) control (without doxycycline), MTB/Cat^TMILA bigenic (without doxycycline), and MTB/Cat^TMILA bigenic mice on food and water supplemented with doxycycline for 1-month respectively. Using antibodies to full-length β-catenin and the myc-epitope tag, the transgene-derived ΔN89β-catenin protein (75kDa) is only detected in the MTB/Cat^TMILA bigenic treated with doxycycline (lane 3); β-actin serves as a loading control. Each lane represents a protein isolate pooled from four individual mice per genotype and treatment.