Journal: Molecular Therapy. Nucleic Acids
Article Title: The Antibiotic-free pFAR4 Vector Paired with the Sleeping Beauty Transposon System Mediates Efficient Transgene Delivery in Human Cells
Figure Lengend Snippet: Analysis of Transfection Efficiency, PEDF Expression, and Secretion by Primary Human RPE Cells 13 and 21 Days after Transfection (A) Eight individual cultures of RPE cells isolated from 4 donor eyes (age, 74.0 ± 6.0 years; gender, 2 males and 2 females; time postmortem, 26.3 ± 3.9 hr; cultivation time before transfection, 48.8 ± 10.0 days) were transfected with 30 ng pFAR4-CMV SB100X SV40 transposase and either 470 ng pT2-ITRs CAGGS Venus (0.11 pmol) or 324 ng pFAR4-ITRs CAGGS Venus (0.11 pmol + 146 ng pFAR4/empty plasmid DNA) or without plasmid DNA as a control. Venus expression for 1 × 10 5 cells was analyzed by flow cytometry at 13.0 ± 1.8 days after transfection. Statistical analysis using an unpaired two-tailed t test showed a significantly greater number of fluorescent cells when transfected with pFAR4-Venus than when transfected with pT2-Venus (p = 0.0277). However, no difference in mean fluorescence intensity was noted. (B) PEDF secretion was analyzed by western blots of culture medium of 1 × 10 4 RPE cells isolated from 10 human donor eyes (age, 68.3 ± 14.8 years; gender, 6 males and 4 females; time postmortem, 29.5 ± 19.6 hr; cultivation time before transfection, 39.3 ± 22.3 days) and transfected with equimolar concentrations of pT2-PEDF (470 ng) or pFAR4-PEDF (313 ng + 157 ng pFAR4/empty plasmid DNA) in the presence of 30 ng of SB100X carried by pFAR4 or without plasmid DNA as a control. Culture supernatants of 72 individual control cultures, 116 individual pT2-PEDF cultures, and 114 individual pFAR4-PEDF cultures were analyzed for total PEDF secretion 21.1 ± 1.1 days after transfection. Loading of equal amounts of culture supernatants was proven by SDS-PAGE Coomassie G-250 staining. Using anti-PEDF antibodies, signal intensities obtained with cells transfected with either pFAR4-PEDF or pT2-PEDF were normalized to the signal intensities obtained with control cells electroporated without plasmid DNA. Total PEDF secretion from pT2-PEDF- or pFAR4-PEDF-transfected cells was significantly higher than from cells transfected without plasmid DNA (p ≤ 0.001), and pFAR4-PEDF-transfected cells secreted a significantly higher PEDF level than pT2-PEDF-transfected cells (p ≤ 0.001, one-way ANOVA with Tukey’s multiple comparisons test). (C) Relative PEDF gene expression was analyzed by qPCR for 1 × 10 4 RPE cells isolated from 7 donor eyes (age, 63.7 ± 15.5 years; gender, 5 males and 2 females; time postmortem, 32.1 ± 23.4 hr; cultivation time before transfection, 37.6 ± 17.8 days), transfected with equimolar concentrations of pT2-PEDF and pFAR4-PEDF (using the same conditions as described above), and cultured for 21.3 ± 1.3 days after transfection. Data for total (endogenous + recombinant) PEDF gene expression are presented as a box and whisker plot (whiskers, minimum to maximum). Total PEDF gene expression in pFAR4-PEDF-transfected cells was related to that obtained with pT2-PEDF-transfected cells, which was set to 1 (dashed line). The difference is statistically significant, p = 0.0406 (one sample t test), exhibiting a 1.89-fold increase. Transfection of RPE cells with pFAR4-PEDF allowed for a significant increase in total PEDF expression (p = 0.0333, unpaired two-tailed t test) compared with the endogenous PEDF level (data not shown). (D) Total PEDF secretion for 1 × 10 4 cells transfected with equimolar concentrations of pT2-PEDF and pFAR4-PEDF (using the same conditions as described in B) was quantified by ELISA using RPE cells isolated from 9 human donor eyes (age, 67.4 ± 15.4 years; gender, 6 males and 3 females; time postmortem, 30.1 ± 20.7 hours; cultivation time before transfection, 41.6 ± 22.5 days). Culture supernatants of 36 individual control cultures, 32 individual pT2-PEDF cultures, and 32 individual pFAR4-PEDF cultures were analyzed 21.0 ± 1.1 days after transfection. Total PEDF secretion of pT2-PEDF and pFAR4-PEDF transfected cells was compared with non-transfected control cells (p = not significant for pT2-PEDF-transfected cells and p ≤ 0.001 for pFAR4-PEDF-transfected cells), and total PEDF secretion of pT2-PEDF-transfected cells was compared with total PEDF secretion of pFAR4-PEDF-transfected cells (p ≤ 0.05, one-way ANOVA with Tukey’s multiple comparisons test). All data are expressed as mean ± SD.
Article Snippet: Plasmid Constructs and Manipulation pFAR4-ITRs is a derivative of the antibiotic-free pFAR4 vector that contains the ITR sequence elements extracted from pT2/BH (a gift from P. Hackett, Addgene plasmid 26556; Cambridge MA, USA). pFAR4-ITRs-SV-Neo (3,385 bp), pFAR4-ITRs-CAGGS-Venus (pFAR-Venus, 4,227 bp), and pFAR4-CMV-SB100X (3,094 bp) were constructed using standard cloning procedures and the following donor templates: pT2/SV-Neo (5,155 bp), pT2-CAGGS-Venus (pT2-Venus, 6,132 bp), and pCMV-CAT(T7)-SB100X (4,752 bp).
Techniques: Transfection, Expressing, Isolation, Plasmid Preparation, Flow Cytometry, Cytometry, Two Tailed Test, Fluorescence, Western Blot, SDS Page, Staining, Real-time Polymerase Chain Reaction, Cell Culture, Recombinant, Whisker Assay, Enzyme-linked Immunosorbent Assay