Efficient Non-Viral Integration and Stable Gene Expression in Multipotent Adult Progenitor Cells
Figure 2
Stable expression mediated by SB transposase and C31integrase in cultured human fibroblasts. (a) Schematic diagram of plasmids cotransfected into HT1080 cells in a colony-forming assay. The integrating vector (pKT2/NAG) is described in Figure 1. Recombinase-encoding plasmids are transcriptionally regulated by the cytomegalovirus immediate early promoter (CMV). The firefly luciferase encoding expression vector (Luc) serves as a control for colony formation resulting from random recombination compared to stable gene transfer mediated by SB transposase (SB) or C31 integrase (Int). (b) Colony-forming assay for integration efficiency. HT1080 cells (4-5 × 105) were cotransfected in triplicate with pKT2/NAG (500 ng) and 150 ng, 500 ng, or 1500 ng of Luc, SB, or Int encoding expression plasmids as described in Section 2. The number of G418-resistant colonies per 3 × 104 cells plated is shown for each group (). Values are reported as mean ± SE. (c) GFP expression in transgenic HT1080 cells. Examples of flow cytometry plots are shown for unmanipulated HT1080 cells (left panel; GFP negative) or an expanded G418-resistant clone (right panel; GFP positive). (d) Percentage GFP-positive cells determined by flow cytometric analysis of 10 independent G418-resistant clones expanded from HT1080 cells cotransfected with pKT2/NAG and 500 ng (open circle) or 1500 ng (filled circle) of Luc, SB, or Int expression vectors. Mean percentages of GFP-positive cells for each condition are indicated by solid lines.
