Efficient Non-Viral Integration and Stable Gene Expression in Multipotent Adult Progenitor Cells
Figure 5
Stability of gene expression in MAPC. (a) Percentage GFP positive cells determined by flow cytometric analysis of 24 independent G418-resistant clones expanded from MAPC cotransfected with pKT2/NAG and Luc, SB, or Int expression vectors. Mean percentages of GFP+ cells for each condition are indicated by black bars. (b) Southern hybridization analysis of SB- and C31-mediated integration events. Top shows a schematic representation of the 6709 bp circular plasmid nucleofected into rat MAPC and the location of the GFP probe. See legend of Figure 1 for explanation of sequence elements contained in the vector. SpeI and BamHI restriction sites were used for digestion of high molecular weight genomic DNA isolated from each G418-resistant clone. The SpeI site is located 2.4âkb from the breakpoint in the attB site. The BamHI site is located 3.5âkb from the right IR/DR. The bottom figure shows the hybridization image of genomic DNA digested with SpeI (C31) and BamHI (SB) hybridized with the GFP probe. Clone number is indicated across the top, and marker positions are indicated along the sides. (c) Flow cytometry of representative stable G418-resistant clones exhibiting GFP expression in HT1080 human fibroblasts and rat multipotent adult progenitor cells (rMAPCs) with integrating vector alone (left panel), integrating vector plus C31 integrase (middle panel) and integrating vector plus SB transposase (right panel) compared to mock transfected cells of each type (black). (d) FACS analysis of individual clones was also measured and the average %GFP+ cells determined. Bar graph showing the average GFP% expression in HT12080 (dark bars) and rMAPC (light bars). Error bars represent standard deviation.
