FIGURE 1

Engineered zinc finger proteins (ZFPs) activate Pedf expression. (a) The expression plasmid for ZFP-hPEDF or empty vector control was transfected into HEK293 cells in duplicate; 72 hours after transfection, total RNA was isolated, and the level of Pedf messenger RNA (mRNA) and 18S RNA were measured by real time reverse transcriptase polymerase chain reaction (RT-PCR) (Taqman), the Pedf/18S ratio was used for normalizing Pedf expression. The Pedf/18S ratio of cells transfected with empty vector control was set as 1. The bars represent the mean (SD) of PEDF/18S ratio of duplicate transfections, each measured by duplicate RT-PCRs. (b) The same as a, except that ARPE-19 cells were transfected. (c) Seventy-two hours after transfection into HEK293 or ARPE-19 cells, culture media were collected and analyzed by Western blotting with an anti-PEDF polyclonal antibody. The cell numbers at the time of harvesting were not significantly affected by the transfection of either the ZFP-hPEDF or the empty vector, as verified by cell counting and the levels of 18S RNA (RT-PCR). Recombinant PEDF was used as a positive control. (d) The same as a, except that U87MG cells were transfected. Because the basal level of PEDF mRNA was near the lower detection limit of RT-PCR, the ratio of PEDF/18S ratio of cells transfected with ZFP-hPEDF was set as 1. (e) The expression plasmid for ZFP-mPEDF and empty vector control were transfected into Neuro2A cells in duplicate; RNA analysis was done as in a. (f) Neuro2A cells were transfected as described earlier, and after 48 hours the media were replaced. Replicates of transfected cells were maintained under normoxic (20% O₂) or hypoxic (0.5% O₂) conditions, and after 24 hours media were collected for Western blots analysis. PEDF, pigment epithelium-derived factor.