Fig. 5

Evaluation of gene stability and gene leakage using various lengths of A2UCOE fragments. A. Design of different A2UCOE fragments. The 1.3 UCOE (1,337 bp) includes the untranslated region (UTR) and exon 1 of HNRNAPA2B1, as well as the promoter-like regions EH38E2541925 and EH38E2541926 extending to include the CBX3 exon 1. The 0.9 UCOE (863 bp) included the part of the HNRNAPA2B1 promoter and extending to exon 1 of CBX3. The 0.7 UCOE (749 bp) contains only the CBX3 exon 1, promoter region, and intergenic sequence. The CBX UCOE (547 bp) includes only the CBX3 promoter region and exon 1. These fragments were incorporated into the FOXN1 Tet-On inducible gene circuit and integrated into the Rogi1 site of PGP1 iPSCs. B. FOXN1 gene leakage in each UCOE group assessed on Day 0 by RT-qPCR (n = 3). C. Flow cytometry analysis of RFP-positive cells in each UCOE group on Day 0 and Day 30 (n = 3). D. Transcription levels of rtTA and mScarlet on Day 0 in each UCOE group by RT-qPCR (n = 3). E. Transcription levels of rtTA and mScarlet on Day 30 in each UCOE group after 30 days of culture without blasticidin by RT-qPCR (n = 3). P-values were calculated using one-way analysis of variance with Tukey’s honestly significant difference test. The data are presented as mean ± SEM. For detailed data, statistical analyses, and exact p-values, see source data file