Figure 5

Acute inactivation of Rb in the developing mouse retina. (A-G) In vivo or in vitro square-wave electroporation and purification of cells with acute Rb inactivation. (C) A retina after electroporation is shown. (D-G) Dissociated cells before and after FACS purification are shown. (H) Real-time PCR analysis of YFP+ purified cells after acute Rb inactivation; each sample was analyzed in duplicate and normalized to Gapdh and Gpi1. (I, J) Immunostaining of purified cells after Rb inactivation. (K-N) To determine if retinal progenitor cells continue to divide after acute Rb inactivation, we scored the proportion of [³H]thy-labeled BrdU+ cells. An example of a double-positive cell is shown in (K). (L) A significant proportion of progenitor cells was sensitive to deregulated proliferation after acute Rb inactivation, as indicated by the increase in double-positive cells. Data are normalized to account for the fraction of cells labeled with BrdU during a 1-h pulse. (M) The proportion of retinal progenitor cells that continue to divide after acute Rb inactivation is shown in the venus-Cre, YFP+ column. (N) Real-time PCR analysis of cell samples used in (L) and (M) revealed a significant increase in the expression of p107 and retinal progenitor cell markers Sfrp1, Erdr1 and Chx10 after acute Rb inactivation. Scale bars: C, D, F, I and J, 10 μm.