From transfection to obtaining a stable cell line takes approximately 5 days to 1 week. However, when including knockout design, primer evaluation, and other preparatory work, the process takes approximately 4 weeks in total in the best-case scenario. It can take longer if the knockout strategy is unsuccessful and requires optimization.

There were two reasons: 

Secreted proteins were considered less likely to be essential genes,  reducing the risk of negatively affecting cell viability. 

Protein secretion consumes cellular energy, so highly secreted proteins were hypothesized to place a greater metabolic burden on cells than intracellular proteins. 

No significant change was observed. The team did not see any differences in cell doubling time following the knockout.

The team did not cultivate cells for six months, so they cannot comment on that timeframe. Their observations are limited to approximately 8 weeks of cultivation. They did not investigate epigenetic changes directly. Instead, they assessed: 

Phenotypic stability 

Transgene copy number 

No specific analyses of epigenetic remodeling were performed. 

Both approaches were used: 

Bulk pools (post-transfection pools) were used during the initial evaluation phase to assess knockout targets. 

Single-cell knockout clones were used during cell line development studies. 

The bulk pools achieved approximately 80% knockout efficiency, whereas the selected knockout clone achieved 100% knockout efficiency. 

Both single and multiple knockouts were tested. 

Single knockouts were evaluated individually. 

Multiple knockouts were also assessed, although those data were not presented during the webinar. 

The multiple knockouts did not provide any additional improvements in productivity or growth. The observed benefits were primarily attributed to a single knockout target that was incorporated into the engineered host cell line, which was identified as the main contributor to the effects shown.