Attenuation of Zinc Finger Nuclease Toxicity by Small-Molecule Regulation of Protein Levels (original) (raw)
Figure 4
Analysis of dd-ZFNs.
Unless otherwise indicated, rates of gene targeting at day 3 were normalized to the rate of gene targeting achieved using 20 ng of the unmodified ZFNs without drug treatment as this was previously determined to be the conditions used to obtain optimal gene targeting with the unmodified ZFNs. The absolute rate of gene targeting using ZFN-1/ZFN-2 at 20 ng in _HEK_293 cells was about 20,000 GFP positive cells per million cells transfected. (A) Titration of transfected DNA of dd-ZFNs in the gene targeting assay with increasing amounts of transfected DNA. (B) Time-course experiment for length of exposure of 1000 nM Shield1 using 5 ng of dd-ZFNs. Hours are given relative to the time of transfection, where “0” is the time of transfection. (C) Drug dose response curve for Shield1 with 5 ng of dd-ZFNs. (D) Gene targeting in the absence and presence of 1000 nM Shield1 for given ZFN pairs at stated DNA concentrations in HEK293 cells. (E) Gene targeting in the absence and presence of 1000 nM Shield1 for given ZFN pairs at stated DNA concentrations in 3T3 cells. The absolute rate of gene targeting using ZFN-1/ZFN-2 at 20 ng in 3T3 cells was about 20,000 GFP positive cells per million cells transfected (2%). (F) Gene targeting in the absence and presence of 1000 nM Shield1 for given ZFN pairs at stated DNA concentrations in HeLa cells per million cells transfected. The absolute rate of gene targeting using ZFN-1/ZFN-2 at 20 ng in HeLa cells was about 2,000 GFP positive cells per million cells transfected (0.2%). (G) Toxicity assay for all iterations of dd-modified and unmodified ZFNs tested in the gene targeting assay relative to Sce. A value of <100% indicates decreased cell survival as compared with Sce, and demonstrates a toxic effect. Statistical analysis was performed using the Student's T-test comparing ZFN-1/ZFN-2 at 20 ng with no drug treatment to dd-modified ZFNs treated with Shield1. “*” indicates a P-value of <.05 and “n.s.” indicates no statistical significance or a P-value of >.05. Error bars are the standard deviation in measurement of three samples.