Figure 4.
One-step generation of cALK2-iPSC from mALK2-hDF by concurrent reprogramming and gene correction. (a) Scheme for cALK2-iPSC generation from mALK2-hDF transfected by the combined mix: reprogramming episomal vectors; single-guided RNA (sgRNA) vector; Cas9-encoding vector; and single-stranded oligodeoxynucleotide (ssODN) template. White dotted circles indicated primary induced pluripotent stem cells (iPSCs) 3 weeks post transfection, these clones were handpicked to 96-well Matrigel-coated plates. (b) Reprogramming efficiency of mALK2-hDF introducing gene-editing tools with reprogramming episomal vectors. Reprogramming efficiency obtained from 2.0 × 105 mALK2-hDF by three independent introduction with sgRNA/Cas9 and reprogramming episomal vectors (gray bar). Reprogramming efficiency for total 88 primary iPSC colonies obtained from 1.0 × 106 mALK2-hDF in a (black bar). (c) Genotyping of the representative iPSC clones driven by concurrent reprogramming and gene correction, performing by Hpy188I digestion, respectively. (d) Sequence analysis of T-vector (pTOP TA V2) clones inserted by the PCR amplicons of homology-directed targeting (HDT)-mediated clones, respectively. The underlined residues indicate the sequences derived from ssODN template. The broken line represents deleted bases. ‘▴' denotes insertion. Scale bar=50 μm (a).