Figure 3

Functional characterization of HPRT1 knock-out cell lines. A: HPRT1 lesions identified in 5 clonal hESC mutant lines. 10 PCR clones were sequenced per cell line. Wild-type sequences shown on top were not seen in any case suggesting that cell clones 1–3 are homozygous. Inserted DNA sequence of clone 5 is given in Additional file 1. B: Quantification of X chromosome copy number in clonal HPRT1 mutant lines using genomic qPCR for an X-linked locus, based on qPCR quantification. Note that all mutant clones cluster with the female parental control. C: Diagnostic PCR for detecting possible random integration of vector sequences into the genome of TALEN-treated cell clones. Analysis was performed at passage 5 after 6-TG selection. Original pTAL7 vectors served as positive control and isogenic parental cells served as negative control. D: PCR and subsequent Cel-1 assay for detection of mutations at the 5 most probably putative TALEN off-target cleavage sites in clonal HPRT1 knock-out cell lines. Genomic DNA from parental hESCs served as control. Note that the additional band in off-target site 1 probably results from a single nucleotide polymorphism and that there are no differences between clonal HPRT1 knock-out cell lines and the parental control. TALEN off-target binding sites are depicted in the right panel with mismatches indicated by asterisks.