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

Figure 5.

Figure 5

Toxicity assay and transplantation of gene-targeted adult fibroblasts. (a) Toxicity of GFP-ZFNs in adult fibroblasts was measured using a fluorescence reporter assay. Cells were transfected with a tdTomato reporter along with an I-SceI, GFP-ZFN, or CCR5-ZFN expression plasmids. ZFN toxicity in cells is reported as fluorescence lost (due to cytotoxic effect) compared to cells transfected with the I-SceI expression plasmid. (b) For transplantation, adult fibroblasts underwent gene correction by nucleofection of 2 µg of each ZFN expression plasmid and 10 µg of donor plasmid. Gene targeting was measured by flow cytometry immediately before transplantation 6 days after nucleofection. Fibroblasts were then injected subcutaneously in a Matrigel matrix. Two weeks after transplantation, the Matrigel plug and surrounding skin were excised, cells dissociated, and plated to allow for fibroblast enrichment from other host-derived infiltrating cell types. On day 6, cells were harvested and analyzed using flow cytometry. ZFN, zinc finger nuclease.