Fig. 4. Effects of RNAi-mediated FLK1 knockdown. (A) A schematic represents the RCAS viral vector (white) encoding the U6 promoter and the CMV-GFP cassette. The shFLK1 RNA (FLK1i) contains a 29 nucleotide antisense (AS, red) and a complementary sense (S, blue) sequence of chicken FLK1 followed by transcription termination sequence (Term). (B-E) Efficiency of FLK1i knockdown in vitro. Fluorescence images show HEK cells co-transfected with the GFP-FLK1 chimeric target and the control U6 (B) or U6-FLK1i (C) plasmid. Western blots (D) show GFP levels in HEK cells co-transfected with the GFP-FLK1 chimeric target and control U6 or U6-FLK1i plasmid. Blots were probed against GFP (top) and AP (bottom), which serves as a transfection efficiency control. Optical densities of GFP signals (E) were normalized against AP signals (n=6; *P<0.03). (F-K) Effects of FLK1i knockdown on RGC development in vivo. Confocal images show retinas infected with the control RCAS (F-H) or RCAS-FLK1i (I-K) viruses at HH stage 17 and immunostained at E6.5 for GFP (F,I), Islet1/2 (G,J) and the respective merged images (H,K). White arrows indicate co-stained cells. Scale bars: 50 µm. (L-N) Quantification of FLK1i knockdown effects in vitro. Explants were electroporated at E5 with the control RCAS or the RCAS-FLK1i construct, and labeled with BrdU for 3 hours before dissociation at 48 hours. Percentages of BrdU⁺ (L), Islet1/2⁺ (M) or NF⁺ (N) cells among transected GFP⁺ cells are shown (n=6; *P<0.03).