Figure 4

Acute inactivation of RB1 in human fetal retinas. (A-D) Representative in situ hybridization of fetal week 16 human retinas with DIG-labeled probes (A, C) and radioactively labeled probes (B, D) shows that RB1 is the major RB family member expressed in proliferating retinal progenitor cells during development. (E-G) Immunofluorescent detection of RB1 (green) in fetal week 16 human retinas confirmed that the protein is expressed in the same pattern as the mRNA. (H) Real-time RT-PCR analysis using TaqMan^® probes for RB1 demonstrated that the expression level does not dramatically change over the course of retinal histogenesis. Data sets were analyzed twice, normalized to Gapdh expression and averaged. (I) The sclera and pigmented epithelium were positive controls for p107 expression. (J) To inactivate RB1 in human fetal retinas, primary tissue was square-wave electroporated with a plasmid that encoded an siRNA to RB1 and a venus-YFP reporter gene. Retinas were then maintained in culture for several days and analyzed for compensation by p107. (K) COS cells transfected with the RB1 SiRNA vector shown in (J) showed a 21-fold reduction in the level of RB1 protein. Densitometry of normalized values for RB1 is shown in the lower portion of panel (K). (L, M) The venus-YFP+ retinal cells that were also [³H]thy+ downregulated RB1 but did not upregulate p107. The negative control SiRNA shown in (M) is the Gapdh SiRNA, but other nonspecific siRNAs gave similar results. The positive control samples are retinas that were square-wave electroporated with a plasmid expressing RB1, p107 or p130 and processed side-by-side with the SiRNA samples. Abbreviations: GCL, ganglion cell layer; inbl, inner neuroblastic layer; onbl, outer neuroblastic layer; PE, pigmented epithelium. Scale bars: G and L, 10 μm; B, D and E, 25 μm.