Fig. 2

PCR analysis of left and right recombination junction and characterization of site-specific integration of seamless vector at LINE-1 in clones. a The table includes the amount and combinations of vector (substrate vector and seamless vector) and Integrase expression vectors transfected in hESCs to establish neomycin resistant F8 knock in clones. b Schematics of left and right junction of the integrated seamless vector in LINE-1. Gel in left panel: PCR analysis showing products of semi-nested PCR obtained with forward primers specific to LINE-1 (F1/F2)/genomic locus (Ch7 1175F, Ch2 1282F, ChX 1093F) and reverse primer (82R) in F8 using template from primary PCR performed using same forward primers and reverse primer (348R) in F8. Gel in right panel: PCR analysis showing products of PCR obtained with forward primer in NeoR gene (Neo 650 F) and reverse primers specific to LINE-1 (R1)/genomic locus (Ch7 440R, Ch2 440R, ChX 831R). Arrows indicate primer position and orientation. Expected PCR amplicon sizes are mentioned for each primer pair at the bottom of each gel. Lanes: L, 1 kb DNA ladder; W, no DNA control; ES, genomic DNA from parental hESCs; F1, F9, B6, B8, genomic DNA from clones. 50 ng of template DNA was used for primary PCRs and 1 μl of primary PCR reaction was used as template for semi-nested PCRs