Establishment of EGFP-stable pool using PTSelect™
As a proof of concept, a stable cell pool expressing EGFP was generated by PTSelect™. PTSelect™-siRNA1 was cloned into the plasmid containing EGFP (Figure 2a) and transfected into CHO-K1 cells by electroporation. On days 3, 7, 10, 17 and 19 after EGFP plasmid transfection, CD4/siRNA1 mRNA and dTomato/ siRNA1 mRNA were introduced by electroporation, followed by depletion. At each depletion, cells in the isolate, supernatant and cells not subjected to depletion (stock) were analyzed by flow cytometry (Figure 2b). The fraction of cells recovered in each round of depletion is shown in Table 1. On day 3, 4.8% of the cells were recovered in the supernatant from 160 x 106 cells; the recovered cells were expanded to 80 x 106 cells. On day 7, the recovery was 30% due to the expansion of enriched cells. However, on day 10, the recovery was only 2.4%, possibly due to the instability of the integrated sequences, resulting in loss from the genome (Würtele et al., 2003). When this population was expanded and enriched on day 17, 80% of the cells were recovered, indicating that the integrated sequences have stabilized in the population. The percentage of recovered cells did not change on day 19, further confirming the stability of the cell population. Thus, it appears that stable cell establishment occurs by day 10, and expansion happens after day 10. Therefore, it is possible to proceed with limited dilution cloning (LDC) on day 10.
The presence of EGFP signals in the supernatant fraction was confirmed by flow cytometry (Supplementary Figure 3). The progressive enrichment of stable cells expressing EGFP in the supernatant was corroborated by a concomitant increase in the number of cells exhibiting decreased dTomato (PE) signal (Figure 2c, top panel). Conversely, the dTomato (PE) signal from the cells in the isolate progressively moved towards the control population signal (Figure 2c, bottom panel). Overall, the results confirm that PTSelect™ can establish a stable cell pool expressing EGFP.