Fig. 4. DsRNA-mediated interference of endogenous Sfrp1 reduces cell differentiation. (A) To assess specificity of the designed dsRNAs, dsRNAs were introduced into MDCK/SFRP1. The ability of Sfrp1 dsRNAs to interfere with myc-SFRP1 protein levels was determined by western blots of the CM of control (con) and dsRNA treated cells using anti-myc antibodies. The Cterm Sfrp1 dsRNA [Cter, two independent evaluations in the upper and lower panels in (A)], and to a lower extent the Nterm Sfrp1 dsRNA (Nter) but not the Gfp dsRNA (Gfp), reduced the protein level in the CM of treated cells. (B) Similar reduction was observed in the mRNA content as determined by RT-PCR analysis. (C) RT-PCR showing chick mRNA levels of Sfrp1 in untreated, control dsRNA (Gfp) or Sfrp1 dsRNA (Cterm) treated retina cells. Cterm Sfrp1 dsRNA induced a decrease in the endogenous level of Sfrp1 mRNA. (B,C) Gapdh amplifications were used as controls. (D) E5 dissociated retina cultures were treated with dsRNAs and cultured for 24 hours. Cultures were immunostained with antibodies against islet1/2, or visinin and counterstained with Hoescht. The presence of Cterm Sfrp1 dsRNA decreases the number of both islet1/2 and visinin-positive cells. *P<0.05; **P<0.001, Student's t-test.