Enrichment of high producers within a clone using PTSelect™
technology
We next tested whether PTSelect™-siRNA activity could be used to enrich
a clone by selecting its high producers. We used an EpoFc-clone (clone
15), which when electroporated with dTomato/ siRNA1 mRNA, showed three
different sub-populations based on their PTSelect™-siRNA1 activity
(Figure 6c, Clone 15-pre-sorted). From this clone, the three distinct
populations were separated. Sorted cells were expanded for four days for
High-PE and Med-PE and two weeks for Low-PE, due to the differing
numbers of sorted cells, and subjected to productivity assay along with
unsorted clone 15. While, there was no significant difference in their
doubling times (Low-PE-22h; Med-PE-20h; High-PE-18h; pre-sorted clone
15-23h), their specific productivities (Low-PE-653; Med-PE-397;
High-PE-0; pre-sorted clone 15-506 mlU/day/cell) correlated with their
ability to suppress the siRNA, as expected. Thus, by enriching clone 15
with cells showing maximum siRNA activity (Low-PE), the productivity of
clone 15 was increased from 506 to 653 mlU/day/cell. The uniformity of
the sorted Low-PE clone was determined (Figure 6c,
15-Low-PE-post-sorted). The cells that exhibit most silencing activity
(100 on the x-axis) had been enriched from 0.2 to
13.2%. Interestingly, clonal drift was also noticed within three weeks
due to the presence of two other cell populations with decreased siRNA
activity (populations 2 and 3). Overall, the results confirm that
PTSelect™ technology can facilitate enrichment of high producers from a
clone, and that it is a sensitive method to monitor single-cell clonal
drift within a cell population.