Fig. 6. A replicating variant of Equalizer-L enables the development of extra-chromosomal cell lines that have stable gene expression with low cell-to-cell variation.

a Over the course of >8 weeks, the Equalizer-L episome produced similar cell-to-cell variation as a chromosomally integrated CMV cassette (CMV cell line) and lower variation than episomes with unregulated promoters. HEK293 cells were used. For all experiments a–c, Equalizer-L was induced with 1-ng/mL doxycycyline. The error bars represent SEM. n = 4 independent trials. Tukey’s multiple comparison test was used to compare the Equalizer-L episome with the other conditions. ns not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. b Representative images of HEK293 cells expressing EGFP from episomes or the chromosome at 23 days post transfection. Each image is displayed with a linear lookup table with the minimum set to 0 and the maximum set to the sum of the mean intensity value and three standard deviations (see Methods). This approach enables a qualitative comparison of the cell-to-cell expression variability despite large differences in mean circuit output. Insets, binary masks to help identify regions of the images that correspond to cells (magenta region). Scale bar, 50 μm. See Supplementary Fig. 18 for images acquired on other days. c The circuit output levels from the Equalizer-L episome were stable for >50 days, whereas episomes with unregulated promoters displayed pronounced declines. Each circuit’s mean output levels were normalized to levels at 9 days post transfection. Induction, sample sizes, error bars, and statistical tests are as in (a). Source data are provided as a Source Data file.