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.