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. 2017 Feb 10;6:302–314. doi: 10.1016/j.omtn.2017.02.002

Figure 4.

Figure 4

Relative PEDF Gene Expression in Primary Human RPE Cells after SB100X-Mediated Transfection

For each donor sample, four control transfections without the addition of miniplasmid DNA and four transfections using 0.03 μg pFAR4-CMV SB100X SV40 transposase and 0.47 μg pFAR4-ITRs CMV PEDF BGH or pFAR4-ITRs CMV PEDFoptimized BGH miniplasmid DNA were performed. Total RNA was isolated 21 days after transfection. Endogenous and total (endogenous + recombinant) PEDF gene expression in PEDF-transfected and PEDFo-transfected cells were related to the PEDF gene expression in control cells, whose expression was set to 1 (dashed line). (A) For 1 × 104 cells, PEDF expression by cells transfected with pFAR-PEDF was analyzed for 25 human donor eyes (age: 65.8 ± 14.2 years; gender: 12 males and 13 females; postmortem time: 32.4 ± 14.5 hr; cultivation time before transfection: 43.0 ± 15.7 days); PEDF expression by cells transfected with pFAR-PEDFo was analyzed for 13 human donor eyes (age: 61.1 ± 16.3 years; gender: seven males and six females; postmortem time: 33.5 ± 16.2 hr; cultivation time before transfection: 39.8 ± 17.1 days). (B) For 5 × 103 cells, PEDF expression by cells transfected with pFAR-PEDF was analyzed for 20 human donor eyes (age: 63.7 ± 14.5 years; gender: 13 males and seven females; postmortem time: 34.6 ± 14.9 hr; cultivation time before transfection: 45.3 ± 16.5 days); PEDF expression for cells transfected with pFAR-PEDFo was analyzed for 13 human donor eyes (age: 61.1 ± 16.3 years; gender: eight males and five females; postmortem time: 32.5 ± 16.1 hr; cultivation time before transfection: 42.6 ± 18.6 days). Data are presented as box-and-whisker plots (whiskers: min to max). Total PEDF gene expression was compared to the endogenous PEDF gene expression (p < 0.0001, unpaired two-tailed t test) and total PEDF gene expression of PEDF-transfected cells was compared to total PEDF gene expression of PEDFo-transfected cells (p = 0.0248 for 1 × 104 cells and p = 0.0161 for 5 × 103 cells, unpaired two-tailed t test).