Fig. 1

Generation of the SETBP1-deficient hESC lines. A Schematic illustration of the wild type (WT) SETBP1 locus and targeting strategy. Exons are shown in black and introns in grey. The three gRNAs targeting exon 4 are indicated in arrows. The homologous arms (HA) corresponding to exon 4 and part of intron 4/5 are indicated in yellow, which are flanked by a PGKpuropA selection cassette in the targeting vector. The positions of the two Nested PCR primer pairs for screening homologous recombination (HR) at the 5′ and 3′ are indicated in black and grey arrows, respectively, with the predicted PCR amplicon size indicated. B Agarose gels showing the final Nested PCR amplicon from the WT and targeted clones (lanes 2, 3, 7); left, 5′ primers; right 3′ primers. C Schematic illustration of the WT and predicted SETBP1 protein product from each of the two SETBP1 mutant allele. Yellow = AT hook domains, blue = SKI homologous region, green = SET binding domain, black = repeat domain. Amino acid sequence alignment of WT SETBP1 protein versus HR and indel allele are shown. Amino acids in red indicate the sequence different from the WT prior to the stop codon. D Representative immunostaining of WT, SETBP1+/- and SETBP1-/- clones for pluripotency markers SOX2 (red), TRA1-81 (green), TRA1-60 (green) and OCT3/4 (red) with DAPI counterstain. Bar graph shows the proportion of EdU⁺ in WT, SETBP1+/- and SETBP1-/- hESCs (P > 0.05). Data presented as mean ± s.e.m of two independent experiments. AA: amino acid; N: NH2 terminal; C:COOH terminal. Scale bar: 50uM