Monitoring clonal productivity by measuring the silencing activity
of PTSelect™-siRNA
To demonstrate that PTSelect™-siRNA can monitor clonal stability, we
selected three clones previously subjected to stability assay (Figure
3d, Clone ES4, Clone ES21 and ES24). Clones ES4 and ES21 passed the
stability test while clone ES24 failed. At selected times during the
stability study (0, 4, 8 and 12 weeks), cells were frozen. Samples from
all four time points from each of the three cell lines were transfected
with dTomato/siRNA1 mRNA and analyzed by flow cytometry. Notably, both
clones that passed the productivity test (Clones ES4 and ES21)
maintained a significant number of cells at 100 on the
x-axis (i.e., with little to no dTomato (PE) fluorescence) through week
12, indicating the presence of cells with high GOI expression. This cell
population with negligible dTomato expression is absent in the clone
that failed the productivity test (Figure 6b). These results further
validate the correlation between the PTSelect™-siRNA activity and GOI
expression and demonstrate that the PTSelect™-siRNA silencing activity
can be utilized as a marker to monitor the productivity of
PTSelect™-generated clones.