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. 2010 Apr 13;18(6):1103–1110. doi: 10.1038/mt.2010.57

Figure 2.

Figure 2

Gene targeting in ES cells. (a) Titration of donor plasmid and ZFNs in ES cells. Cells were transfected using Lipofectamine 2000 with different amounts of donor and ZFN plasmids. From left to right, amounts of donor plasmid increased, whereas ZFN amounts decreased. Transfection was performed with Lipofectamine 2000, and the amounts of donor plasmid and ZFNs in each lane are indicated as donor (ng), ZFN1 (ng)/ZFN2 (ng). (1) 100, 350/350; (2) 400, 200/200; (3) 600, 100/100; (4) 700, 50/50; (5) 750, 25/25; (6) 775, 13/13. Fifteen hours after transfection, media were changed to normal ESLX. Gene-targeting events were analyzed 4 days after transfection using flow cytometry. (b) Gene targeting in ES cells using two sets of GFP-ZFNs, with and without vinblastine treatment. Cells were plated in ESLX with and without vinblastine (100 nmol/l). Transfection mix was added, and 15 hours later, removed and replated with ESLX. Gene-targeting events were analyzed as in a. 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. ES, embryonic stem; ZFN, zinc finger nuclease.