Use of antibiotic‐resistance‐free plasmids for non‐viral transfection of pigment epithelium‐derived factor in rat primary cells

Abstract

AbstractPurpose We aim to assess the pigment epithelium‐derived factor (PEDF) production in cells after transfection with plasmids free of antibiotic resistance markers (pFAR4) in a non‐viral Sleeping Beauty transposon system (SB100X).Methods ARPE‐19 cell line and primary rat iris and retinal pigment epithelial (IPE and RPE) cells were transfected with SB100X transposase and PEDF transposon encoding pFAR4 plasmids (1:16 ratio). Control cells were transfected with SB100X and pFAR4‐Venus plasmids. Supernatants were taken every 24 hours for kinetics. The effect of transfecting different amount of cells was also analyzed. PEDF production was tested by Western blot and ELISA.Results A cumulative PEDF secretion over time in ARPE‐19 and primary cells was shown, reaching almost 3‐fold higher PEDF production in ARPE‐19 cells. Control cells did not express recombinant PEDF, but showed fluorescence. The transfection of 10,000 cells clearly showed higher PEDF expression than cells that were transfected as a group of 70,000 and subsequently divided.Conclusion Rat primary cells are efficiently transfected and produced PEDF as it happens in ARPE‐19 cell line. Merging of the SB100X and pFAR4 technologies led to increased PEDF expression and improved safety by avoiding the transfer and potential integration of antibiotic resistance genes. This study demonstrates the feasibility of our approach based on ex vivo transfection of a low number of primary autologous pigment epithelial cells.