Journal: Investigative ophthalmology & visual science

Article Title: Sleeping Beauty transposon-mediated transfection of retinal and iris pigment epithelial cells.

doi: 10.1167/iovs.12-9951

Figure Lengend Snippet: FIGURE 2. Optimization of transfection efficiency in ARPE-19 cells by varying the ratio of SB100X transposase expression plasmid to pT2-CAGGS- Venus transposon plasmid. (A) Cytofluorimetric analysis of cells transfected with increasing ratios of SB100X transposase to pT2-CAGGS-Venus transposon plasmid 8 days after electroporation. (B) Continuous cytofluorimetric analysis of transfected cells showed stable Venus expression followed for longer than 16 months in culture. Transfected cells were passaged weekly and flow cytometric analysis was performed every other week. Phase contrast and fluorescence micrographs, taken 425 days after transfection, demonstrated consistent Venus expression and epithelial morphology. (C) SDS-PAGE and Western blot analysis of cell lysates show the levels of intracellular Venus protein and SB100X transposase in pT2-CAGGS-Venus transfected cells. Venus protein (~27 kDa) was visualized by SDS-PAGE Coomassie G-250 staining. SB100X (~37 kDa) was detected using anti-Sleeping Beauty transposase antibodies. Note that the band intensity of GAPDH (~36 kDa) is similar in all lanes, indicating loading of equal amounts of protein.

Article Snippet: Blots blocked with 3% BSA/tris-buffered saline (TBS) overnight at 48C were incubated for 2 hours at room temperature with anti-Penta-His antibodies (mouse monoclonal, 1:500; Qiagen) diluted in 3% BSA/TBS for the detection of recombinant PEDF, with anti-GAPDH antibodies (mouse monoclonal, 1:5000; Novus Biologicals, Littleton, CO) for the detection of GAPDH, and with anti-Sleeping Beauty transposase antibodies (goat polyclonal, 1:1000; R&D Systems, Minneapolis, MN) for the detection of SB100X transposase.

Techniques: Transfection, Expressing, Plasmid Preparation, Electroporation, SDS Page, Western Blot, Staining

Journal: Investigative ophthalmology & visual science

Article Title: Sleeping Beauty transposon-mediated transfection of retinal and iris pigment epithelial cells.

doi: 10.1167/iovs.12-9951

Figure Lengend Snippet: FIGURE 3. Optimization of transposition-mediated PEDF gene delivery in ARPE-19 cells by varying the ratio of SB100X transposase expression plasmid to PEDF transposon plasmid. (A) Cells, transfected with increasing SB100X to pT2-CMV-PEDF/EGFP transposon plasmid ratios, and their culture media were analyzed 8 days after electroporation by cytofluorimetric analysis for EGFP expression and by Western blot analysis for secretion of recombinant PEDF. (B) Continuous flow cytometric analysis of pT2-CMV-PEDF/EGFP and pT2-CAGGS-PEDF/EGFP transfected cells (ratio 1:32) showed an increased percentage of fluorescent cells within the population (open diamonds and triangles, respectively). Using cell sorting, it was possible to obtain populations of ‡99% transfected cells (filled diamonds and triangles, respectively) that exhibited stable fluorescence for more than 16 months that the cells have been in culture. Transfected cells were passaged weekly and cytofluorimetric analysis was done every other week.

Article Snippet: Blots blocked with 3% BSA/tris-buffered saline (TBS) overnight at 48C were incubated for 2 hours at room temperature with anti-Penta-His antibodies (mouse monoclonal, 1:500; Qiagen) diluted in 3% BSA/TBS for the detection of recombinant PEDF, with anti-GAPDH antibodies (mouse monoclonal, 1:5000; Novus Biologicals, Littleton, CO) for the detection of GAPDH, and with anti-Sleeping Beauty transposase antibodies (goat polyclonal, 1:1000; R&D Systems, Minneapolis, MN) for the detection of SB100X transposase.

Techniques: Expressing, Plasmid Preparation, Transfection, Electroporation, Western Blot, Recombinant, FACS

Journal: Investigative ophthalmology & visual science

Article Title: Sleeping Beauty transposon-mediated transfection of retinal and iris pigment epithelial cells.

doi: 10.1167/iovs.12-9951

Figure Lengend Snippet: FIGURE 6. Analysis of SB100X-mediated transposition in primary bovine RPE and IPE cells. (A) Western blot analysis of rPEDF secretion by primary bovine RPE cells transfected with varying ratios of SB100X transposase expression plasmid to pT2-CMV-PEDF/EGFP transposon plasmid. Primary bovine RPE (B) and IPE (E) cells were co-transfected with SB100X transposase and either pT2-CAGGS-Venus or pT2-CMV-PEDF/EGFP transposon plasmid at a ratio of 1:20. Both transfected RPE (C) and IPE (F) cells showed intense and stable fluorescence for the 150 days the cells have been cultured. Note that the cells retain the typical cobblestone morphology of epithelial cells. (D, G) Western blot analysis demonstrated sustained rPEDF secretion for the 200 days the cells have been in culture. Recombinant PEDF (~48 kDa) was detected using anti-Penta-His antibodies.

Article Snippet: Blots blocked with 3% BSA/tris-buffered saline (TBS) overnight at 48C were incubated for 2 hours at room temperature with anti-Penta-His antibodies (mouse monoclonal, 1:500; Qiagen) diluted in 3% BSA/TBS for the detection of recombinant PEDF, with anti-GAPDH antibodies (mouse monoclonal, 1:5000; Novus Biologicals, Littleton, CO) for the detection of GAPDH, and with anti-Sleeping Beauty transposase antibodies (goat polyclonal, 1:1000; R&D Systems, Minneapolis, MN) for the detection of SB100X transposase.

Techniques: Western Blot, Transfection, Expressing, Plasmid Preparation, Cell Culture, Recombinant