Improved mutagenesis and universal applicability of the pTAL7 vector system. A: Cel-1 assay following transfection of 293 T and hESCs with HPRT1 talen pair #2 in different expression vector backbones. B: Functional confirmation of results in (A) by 6-TG selection. C: Quantification of functional mutations in HPRT1 following 6-TG selection, based on scoring of colony numbers. For the control without antibiotics and the pTAL4 vectors, no puromycin/blasticidin selection was performed. Note the strong increase in mutation rates upon pre-selection for double-transfected cells using transient administration of antibiotics. Error bars: SEM (n = 4). D: Applicability of the pTAL7 system to alternative genomic loci as shown by Cel-1 assay (left panel) or restriction digestion (right panel: NHEJ is indicated by enrichment of undigested fragments). hESCs were transfected with either a GFP control vector or the respective TALEN vectors, followed by subsequent gPCR amplification of TALEN target regions. E: Restriction digestion and characterization of 19 clones expanded after TALEN transfection without 6-TG selection. Except in panel C, pTAL7-transfected cells were routinely enriched by transient puromycin/blasticidin administration.