Fig. 5

Transgene expression from the weak heterologous promoter is consistent and homogeneous in the potential GSH loci of isogenous cell clones. Clonally-expanded isogenous cells harboring the ∆CMV-driven EGFP in the potential GSH loci were able to consistently express the transgene. A-a, B-a) Schematic depiction of the process in which clonally-isolated cells were cultured for about six months. Offset (A-b, B-b for cROSA; A-e, B-e for cHIPP; A-h, B-h for cOVA at the end of MTH4 and MTH6) and overlay (A-c, B-c for cROSA; A-f, B-f for cHIPP; A-i, B-i for cOVA at the end of MTH4 and MTH6) illustrate the EGFP expression levels for correctly-targeted isogenous cell clones targeted at the cROSA (clones R2, R5, R8), cHIPP (clones H1, H4, H6), and cOVA (clones O3, O5, O8) loci. Shifting the peak to the right in the offsets shows an increase in the expression of EGFP (arrows show the high density of EGFP-positive cell clones). The MFI index in the cROSA (A-d, B-d) and cHIPP (A-g, B-g) clones with cOVA clones at the end of MTH4 and MTH6 were compared. Green squares show the average expression of EGFP for cROSA, cHIPP, and cOVA clones in the green channel. No expression signal was detected in the red channel (red square). The integrated density (ID) index was compared using the ImageJ (A-j, B-j) and GNUastro (A-k, B-k) software. The copy number of EGFP transcripts (A-l, B-l), and the expression levels of EGFP (A-m, B-m) were determined in the main experimental groups (i.e., cROSA and cHIPP) versus the control group (cOVA) at the end of MTH4 and MTH6. ns: non-significant, ***: p < 0.005, and ****: p < 0.0001 are statistically significant. Avg.: The average expression of EGFP. N: number. Integrated density by imageJ (ID by Im.J). Integrated density by GNUastro (ID by Gnu). Copy Number (CN) by qPCR. Expression by Western Blotting (Exp. by WB)