Figure 2.

Generation and Validation Strategy for CRISPR/Cas9-Mediated Knockin in hAMSCs

(A) FACS analysis of EGFP expression level following co-transfection by electroporation of pCRISPR-Cas9/CD4-TK2 and pMA-DONR-EF1α-EGFP-tTK. EGFP expression in hAMSCs decreased progressively until stabilization. (B) EGFP-expressing hAMSCs (0.14% from the total population) were sorted at day 22 post-transfection and expanded. (C) Bright-field images showing control and sorted hAMSCs. Scale bars, 20 μm. (D) Schematic representation of the TK2 exon 5 after integration and predicted amplicons for validation of integration; arrows indicate the hybridization site for forward and reverse primers flanking 5′ and 3′ insert junctions at exon 5. (E) Agarose gel showing the size of predicted PCR amplicons from amplification of genomic DNA extracted from sorted and expanded hAMSCs after CRISPR/Cas9 modification.