Generation of the conditional Cdc42 overexpression mouse mammary gland model. (A) Schematic model of TetO-Cdc42/MMTV-rtTA bigenic mice. The TetO-Cdc42 transgene contains the tetracycline operator and minimal cytomegalovirus (CMV) promoter with human Cdc42 and IRES luciferase immediately downstream. TetO-Cdc42 mice were bred with the MMTV-rtTA mice to drive transgene expression in the mammary gland . In the presence of dox, rtTA activates expression of the Cdc42 transgene. Dox-treated MMTV-rtTA mice serve as controls for all studies. (B) Graph depicts average luciferase levels of control (MMTV-rtTA, + dox) and lines 3 and 4 bigenic (TetO-Cdc42/MMTV-rtTA, -/+ dox) mammary glands (n = 15,1,1,10,14). Transgene is induced only in bigenic mice treated with dox. (C) Western blot analysis of Cdc42 expression on five pooled whole gland lysates per group after 3 weeks (Line 3) and 1 week (Line 4) of dox treatment. Densitometry values are normalized to loading control and displayed as fold change compared to control. (D) Graph shows luciferase levels of mammary organoids and stromal cells isolated from Cdc42 overexpressing and control mice (n = 3 animals pooled per group). Cdc42, cell division cycle 42; dox, doxycycline; MMTV-rtTA, mouse mammary tumor virus reverse tetracycline transactivator; TetO, tetracycline operator.