Figure 4.
Modifications at the human IL2RG locus. (a) Target sites in human IL2RG gene. Target sites are highlighted by gray shaded (TALEN) or black (ZFN) boxes, respectively. The RVDs of the engineered TALEs as well as the expected target sequences are indicated. (b) Comparison of the activities and toxicities of IL2RG-specific designer nucleases. HEK293T cells were co-transfected with expression plasmids-encoding IL2RG-specific TALEN and ZFN, respectively, and a reporter plasmid harboring an IL2RG-dsEGFP fusion gene. The graphs display reduction of the mean fluorescent intensity (MFI) of IL2RG-dsEGFP (left) and relative cell survival as compared to cells expressing a nonfunctional nuclease indicated with ‘–’ (right). NH and NC designate the TALEN scaffold used. Statistically significant differences in activity and toxicity between the TALEN and ZFN tested are indicated by *(P < 0.05) or **(P < 0.01), respectively. (c) Disruption of endogenous human IL2RG locus. After transfection with TALEN (NC scaffold) and ZFN expression vectors, genomic DNA was extracted and used as a template for PCR amplification. Amplicons encompassing the target sites were digested with the mismatch-sensitive T7 endonuclease 1 (T7E1). Arrows indicate the expected positions of the T7E1 digestion products. Numbers at the bottom designate the average percentage of modified alleles (n = 2). TALENs targeting the CCR5 locus (C5-TALEN) were used as negative controls. L and R refer to the nuclease subunits, binding to the left or right target half-sites, respectively.