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.