Figure 4.

ZFN-mediated gene targeting in primary cells. (a) Gene targeting in homozygous and heterozygous ROSA-3T3s: transfections were performed using Lipofectamine 2000 with the indicated amounts of donor plasmid and ZFNs. The next day, media were changed, and on day 4, gene-targeting events were analyzed. (b,c) Gene targeting in MAF/MEFs: transfection of plasmids was performed by nucleofection using 2 µg of each ZFN and the indicated amounts of donor plasmid. Gene-targeting events were analyzed 4 days after transfection. (d) Gene targeting in astrocytes: targeting was performed in the same manner as MAFs/MEFs. In the upper left of each graph is a representative flow cytometry plot after targeting in which GFP fluorescence is measured in the y axis and background orange fluorescence along the x axis. The number in the left corner of the flow plot is the percentage of GFP⁺ cells. The corrected ROSA-3T3 cells show much higher GFP fluorescence than the primary cells demonstrating that although the ROSA26 locus is ubiquitously expressed, expression levels from the locus vary significantly depending on the cell type. Data are presented as mean ± SEM (*P < 0.05). ZFN, zinc finger nuclease.