Efficient generation of Doxycyline-inducible stable cell lines free of background expression.

<p>(a) Knock out <i>Maged1</i> Mouse Embryonic Fibroblasts (MEFs) were stably transduced with LVTPTIP-3xFlag-MAGED1b and treated with 1μg/ml Doxycycline for 4, 8 or 24hrs. Whole cell extracts were analysed for MAGED1b (anti-Flag) and tubulin by immunoblot. (b) LNCaP prostate cancer cells were stably transduced with LVTPTIP-SIM2s-3xFlag or LVTPTIP-SIM2l-3xFlag and treated with 1μg/ml Doxycycline for 4, 8 or 24hrs. Whole cell extracts were analysed for SIM2s or SIM2l (anti-Flag) and tubulin by immunoblot. (c) mRNA of SIM2s and SIM2l from LNCaP prostate cancer cells from (b) was measured by Real-time PCR and normalised to RNA polymerase subunit 2A (POLR2A). Data are mean normalised mRNA fold induction over parent cell line ± SEM of 3 independent experiments. Statistical significance was determined using an unpaired student t-test. n.s. (not significant).