Figure 3.

Western-Blot-Based Quantification of Long-Term PEDF Secretion in Primary Bovine IPE Cells after SB100X-Mediated Transfection

For each IPE sample, two control transfections without the addition of miniplasmid DNA and two 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 carried out. Culture supernatants were analyzed for total PEDF secretion 7, 15, 19, 32, and 52 weeks after transfection. Western blot signal intensities of PEDF-transfected and PEDFo-transfected cells were normalized to the signal intensities of the control cells. Data are presented as mean ± SD. Mean signal intensities of PEDF secretion 21 days after transfection are indicated by the dashed line. For 365 ± 3 days, levels of PEDF secreted by pFAR-PEDF- and pFAR-PEDFo-transfected cells were analyzed for (A) 1 × 10⁴ cells and (B) 5 × 10³ cells. Statistical analysis using an unpaired two-tailed t test showed a significant difference between transfected cells and non-transfected control cells. No significant difference was observed between cells transfected with pFAR-PEDF and pFAR-PEDFo at any time period.