Figure 3. Generation of a transgenic podocyte-specific hM₃D mouse model.

(A) Schematic of the ROSA26 locus with the FLAG.hM₃D sequence before and after Cre recombination. Triangles indicate loxP sites and rectangles indicate exons. (B) Southern Blot analyses were performed with a ROSA26 and a neomycin probe to screen for positive ES-cell clones. (C) Isolated primary glomerular cells were cultured and GFP expression was visualized by confocal microscopy. Glomerular cells from ROSA26^hM3D/wt; PodCre^tg/wt show cytoplasmic GFP expression. ROSA26^wt/wt; PodCre^tg/wt control cells were GFP negative. (scale bar = 50 μm). (D,E) Representative images of Ca²⁺ imaging with Fluo-8 (scale bar = 20 μm). Primary glomerular cells were used for Ca²⁺ imaging with Fluo-8. Cells expressing the receptor showed an instant increase of intracellular Ca²⁺ levels after administration of 100 μM CNO. Control cells did not react to CNO (scale bar = 20 μm). Experiments were performed in three biological replicates. Fluorescence is depicted as F/F_1–5, where the mean value of the first five measurements prior to CNO stimulation was used for normalisation.