Author response image 4. PiggyBAC (PB) integrated constitutive T-REX17 rescue.

(A) Schematic of the ectopic PB T-REX17 construct. Lines indicate randomly integrated sequence context for empty backbone (PB only) or T-REX17 (PB T-REX17). (B) FACS histograms showing percentages of CXCR4⁺ cells during EN differentiation of sgT-REX17 dCas9-KRAB-MeCP2 cells of PB only or PB T-REX17 clones. Clone numbers are indicated. Sample sizes are normalized to 9000 cells /sample. Two independent experiments are displayed. (C) qRT-PCR showing NANOG, SOX17 and T-REX17 expression in PSCs and EN cells of sgT-REX17 dCas9-KRAB-MeCP2 cells integrated for either with PB only or PB T-REX17. Clone numbers are indicated. Fold change is calculated relative to the 18s housekeeping gene. Bar indicate the means, two independent experiments are represented. T-REX17 wild type levels are indicated by the dashed line. Note, ectopic T-REX17 is constantly expressed throughout differentiation.