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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).
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