Generation of podocyte-specific Tsc2 knockout mice, Tsc2Δpodocyte. Homozygous floxed Tsc2 mice were crossed with Nphs2-Cre transgenic mice to generate Tsc2Δpodocyte, Nphs2-Cre+/-; Tsc2flox/flox.
(A) PCR genotyping of genomic DNA from mouse tails. NPHS2-Cre+/-, Tsc2wt/wt mice (Nphs2-Cre) and NPHS2-Cre-/-, Tsc2flox/flox mice (Tsc2flox/flox) were used as controls. F, flox; +, wt. (B) Primary cultured podocytes isolated from Tsc2Δpodocyte and wild-type control mice at 4 weeks of age demonstrated that Tsc2 mRNA was knocked down by over 80% in the podocytes of Tsc2Δpodocyte mice compared with control mice. Results are expressed as means ± s.d. *P < 0.05 compared with age-matched controls. (C) Western blot analysis of primary cultured podocytes isolated from Tsc2Δpodocyte and control mice. TSC2 gives a band at 200 kDa (arrow). Non-specific bands were also detected above the TSC2 band in all samples. β-Tubulin was used as an internal control. (D) Tsc2 mRNA expression in various tissues of Tsc2Δpodocyte and wild-type controls. Expression levels of Tsc2 mRNA in Tsc2Δpodocyte were comparable with those in controls in all the tissues examined, including the renal cortex. White bar, Nphs2-Cre; dotted bar, Tsc2flox/flox; black bar, Tsc2Δpodocyte.