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. 2008 May 29;36(12):3926–3938. doi: 10.1093/nar/gkn313

Figure 6.

Figure 6.

Stable shift in mitochondrial heteroplasmy in cells expressing mutation-specific ZFNs. The m.8993T>G mutation-specific ZFNs, F-NARPd-Fok or F-NARPd-Fok-L35-Fok, were co-expressed with eGFP from a single vector in heteroplasmic cybrid cells containing ∼85% of mtDNA molecules with the NARP m.8993T>G mutation (dashed line). Two days after the transfection cells were FACS sorted using eGFP as a marker, then the sorted cells were grown on non-selective medium for a further 28 days. The percentage of mutant mtDNA was assessed again at 2 and 30 days. Mock indicates cells transfected with vector alone. Symbols: *P < 0.05; **P < 0.01 in two-tailed t-test, unequal variance. (A) Schematic illustration of the experimental approaches taken. (B) Differences in the proportion of the wt and mutant mtDNA in bulk population of transfected cells at 2 and 30 days. The difference between mock and F-NARPd-Fok-L35-Fok transfected cells after 30 days is highly significant (P = 0.007), whereas the difference between mock and the F-NARPd-Fok monomer is not significant (P = 0.54). (C) Total mtDNA copy number in the transfected cells measured at 2 and 30 days expressed in arbitrary units (a.u.). The results are normalized to mock transfected cells. (D) Differences in the proportion of the wt and mutant mtDNA in individual clones, randomly picked at 30 days post-transfection. The red horizontal bar indicates an average value for each construct. The difference between mock and F-NARPd-Fok-L35-Fok transfected cells after 30 days is highly significant (P = 0.003), the difference between mock and the F-NARPd-Fok monomer is of much lower significance (P = 0.4).