Targeting strategy using genetically engineered nucleases to generate OCT4-eGFP-2A-Puro hESC lines.

<p>(A) The structure of Xanthomonas sp TALE protein. Each nucleotide-binding module is comprised of a 34 amino acid sequence, inside of which is embedded one of 4 repeat variable domains (RVD). The sequence of this di-amino acid RVD dictates the deoxynucleotide-binding cipher: NG is highly specific for deoxythymidine, HD for deoxycytidine, NI for deoxyadenosine, and NH for deoxyguanosine. (B) Schematic overview of the targeting strategy using TALENs to knock eGFP onto the OCT4 coding sequence. Red line represents the stop codon. Regions where genotyping PCR primer pairs bind are highlighted for 5′F, 5′R, 3′F and 3′R. (C) Genotyping PCR for i) 5′ arm of insertion using primers 5′F and 5′R (1180 bp) ii) 3′ arm of insertion using primers 3′F and 3′R (1000 bp) iii) endogenous allele using primers 5′F and 3′R for parent CyT49 line and three of the generated knock-in lines OCT4-2, OCT4-3 and OCT4-28. (D) Schematic overview of the CRISPR-Cas9 targeting strategy. Red line represents the stop codon. A green circle represents the Cas9 endonuclease with the tracRNA in black. The genomic protospacer adjacent motif (PAM) sequence is highlighted in red type and the guide RNA sequence is in bold type. Genotyping PCR primer pairs are the same as for TALEN targeting and are highlighted.