Fig. 2

The 0.9 UCOE enhances long-term gene expression stability in gene-edited iPS cells. A. Location and design of the 0.9 UCOE fragment within the A2UCOE region on human chromosome 7. ATAC-seq data from PGP1 iPSCs show transcriptionally active regions overlapping with a CpG island between the HNRNAPA2B1 and CBX3 genes, suggesting open chromatin in this area. The 0.9 UCOE, isolated from this region, spans 863 bp, including exon 1 of CBX3 and part of the HNRNAPA2B1 promoter. B. Schematic of the all-in-one Tet-On plasmid design with and without the 0.9 UCOE. The 0.9 UCOE was placed upstream of the EF1α promoter to drive stable expression of rtTA and selection markers. C. Experimental workflow for generating and analyzing bulk iPSC populations after gene editing. PGP1 iPSCs were edited using CRISPR/Cas9, followed by 5 days of blasticidin selection. Flow cytometry analysis was performed periodically over the 30-day culture period to monitor the percentage of RFP-positive (mScarlet+) cells. D. Fluorescence microscopy images of the 0.9 UCOE and w/o UCOE bulk populations on Days 0 and 30. E. Quantification of RFP-positive cells by flow cytometry in the 0.9 UCOE and w/o UCOE bulk populations over time (n = 3). F. Reverse Transcriptase Quantitative PCR (RT-qPCR) analysis of transcription levels for rtTA and mScarlet in bulk populations on Day 0 (n = 3). G. RT-qPCR analysis of rtTA and mScarlet transcription levels in bulk populations on Day 30 after 30 days of culture without blasticidin (n = 4). H. RT-qPCR analysis of induced FOXN1 transcription in response to doxycycline on Day 30 after 30 days of culture without blasticidin (n = 4). P-values were calculated using a two-tailed unpaired t-test and one-way analysis of variance with Tukey’s honestly significant difference test. Data are presented as mean ± SEM. For detailed data, statistical analyses, and exact p-values, see source data file