Fig. 4

Generation of T-2A-EGFP knock-in reporter cell line. a Gene targeting strategy of knock-in T-2A-EGFP-PGK-Puro cassette to replace the endogenous T stop codon, facilitated by the CRISPR/Cas9. Gray boxes indicate the exons of the endogenous gene. The arrow indicates the position of the Cas9/sgRNA cut site. The position of the probe for southern blot is indicated. E EcoRI sites. The lengths for each EcoRI-digested genomic DNA fragments are indicated. b Southern blot shows the targeted allele shifted from 4.2 (wild type genomic fragment, WT) to 4.8 kb (knock-in genomic fragment, KI). c Confocal images of SOX2, T, and EGFP immunofluorescence staining over three days of differentiation. Hours of differentiation are indicated. Scale bars = 50 μm. d FACS analysis monitoring the dynamics of the percentage of CXCR4⁺ and T-2A-EGFP⁺ cells over three days of differentiation. The x-axis indicates GFP/FITC channel. The y-axis indicates APC channel