Generation and characterization of inducible MR knock-down mice.

(A) Operating principle of the lentiviral system used for inducible MR knock-down. The vector comprises one cassette consisting of the H1 promoter with tetracycline resistance operator sequences (tetO) and a MR-specific shRNA, and a second one encompassing the tet repressor (TetR) linked to eGFP by a T2A element under the control of the ubiquitin C promoter (Ub-p). In the absence of doxycycline (Dox), the TetR binds to tetO and blocks shRNA expression. After addition of Dox, the TetR is released thus enabling shRNA transcription. Under both conditions, eGFP is expressed constitutively. (B) Peripheral blood leukocytes of one control (con) and one transgenic (tg) mouse were analyzed for eGFP expression by flow cytometry. (C) Kidney, heart, colon, hippocampus, liver, lung, stomach and muscle samples obtained from control (con) and transgenic (tg) mice of line B and D were analyzed for eGFP mRNA expression by RT-QPCR. N = 3–7 (line B), N = 3–5 (line D). Gene expression was normalized to HPRT and is depicted in arbitrary units as mean ± SEM. (D) Kidney sections from one control (con) and one transgenic (tg) mouse of line B were analyzed by immunohistochemistry for eGFP expression. One representative example out of three is shown for each genotype. Size bar: 50 μm.