Highly Effective Ex Vivo Gene Manipulation to Study Kidney Development Using Self-Complementary Adenoassociated Viruses
shRNA-mediated knockdown of WT1 with scAAV2/8 in mk3 cell and cultured kidney. (a) Immunostaining of mk3 (nuclear DAPI, blue, WT1, red) shows that Wt1 expression is repressed in most cells treated with Wt1 shRNA containing scAAV2/8 after 36 hours. (b) This repression was confirmed by western blot. (c) WT1 immunostaining (red) of cultured kidneys after 24 hours shows that WT1 expression is repressed in CM after Wt1 shRNA containing scAAV2/8 treatment. (d) Immunostaining of epithelial maker E-cadherin (red, showing both UB and newly formed nephron tubules, which were marked with a white arrowhead) in cultured kidney after 48 hours. The experiment was tested for three times, with 5 kidney rudiments at each time. (e) The total UB tips and nephron tubules were counted and levels of significance were determined using Student’s t-test. Error bars represent standard errors of the mean. The asterisk indicated a significant difference at .