Fig. 3

Southern blot hybridization of clones targeted with F8 seamless vector. a Schematics of integrated F8 seamless vector at the LINE-1 with information on location of restriction sites within the cassette and in the hESC genome. The Table summarizes the targeted locus and genomic location of seamless vector integration for the clones, based on the genomic fragment sizes. Total genomic DNA from parental hESCs and clones harbouring the complete F8 seamless vector was digested with NsiI and KpnI and subjected to hybridization with DIG-labelled PCR probe complementary to 309 bp in NeoR gene. Bands indicate NeoR gene containing genomic fragments which correlate with the predicted size thereby confirming single copy F8 seamless vector integration at LINE-1. L, 1 kb DNA ladder; ES, genomic DNA from parental hESCs; F1, F9, B6, B8, genomic DNA from clones; + in NsiI Digestion indicates 0.1 ng of linearized substrate vector; + in KpnI Digestion indicates 0.1 ng of NeoR containing KpnI digested fragment (3969 bp) of substrate vector. b An illustration of the location of transgene integration in chromosomes for the targeted clones