FIG. 6.

ZFN-mediated gene targeting in human mesenchymal stromal cells. (a) Experimental setup. UC-MSC.696 cells were seeded in a 24-well plate (50,000 cells/well) 6 h before transduction with 10,000 gc/cell of AAV-ZFN and 2,000 gc/cell of AAV-donor and then treated with indirubin-3′-monoxime. (b) Cell-cycle analysis of UC-MSC.696 cells after treatment with indirubin-3′-monoxime. At 24 h and 48 h after treatment with cytostatics, cells were fixed, stained with propidium iodide, and analyzed by flow cytometry. The percentage of cells in G1 (light gray), S (gray), and G2/M (dark gray) phase is indicated as a bar graph. Asterisks mark a significant difference (p<0.05) from nontreated cells. (c) Gene correction in UC-MSC. The graph shows the numbers of EGFP-positive cells per 100,000 cells measured by flow cytometry. Asterisk indicates a significant increase (p<0.05) over nontreated cells. To verify the presence of faithfully corrected EGFP gene on the genome level, allele-specific PCR was performed. A PCR encompassing a fraction of the PTBP2 gene was used as a control (N=5). I, indirubin-3′-monoxime; M, mock; D, donor; D+Z, donor and ZFN; GT, gene targeting; PTBP2, locus-encoding polypyrimidine-binding protein 2.