Figure 4

TALEN activity in HEK293 cells. A) This schematic shows the general configuration of TALENs that were assembled under the control of a CMV promoter. B) The nucleotide sequences of target sites are 5-TGCCCCTCCACCCCCAC-3’ (top strand), and 5’-TTTCTGTCACCAATCCTG-3’ (bottom stand) flanking a 16-bp spacer. The primers for genomic PCR are 5’-CTTGCTTTCTTTGCCTGGAC-3’ (P1) and 5’-GATCAGTGAAACGCACCAGA-3’ (P2). C) Schematic of the Surveyor nuclease mutation detection assay used to determine TALEN de novo cleavage. D) 2% agarose gel showing the Surveyor nuclease mutation detection result from the assembled AAVS1 TALEN pair. Controls (C1) from mock-transfected HEK293 cells showed no cutting with Surveyor nucleases, whereas cells transfected with the AAVS1 TALEN pair produced expected ~320 bp products. The efficiency was 2.3% and 3.4% at the 24-hour and 48-hour time-points, respectively.