Targeted Gene Knockdown Effects on Recombinant Protein Secretion in CHO Cells

Abstract

Chinese Hamster Ovary (CHO) cells are one of the most widely used cell lines for the production of recombinant therapeutic proteins. In an effort to better understand the molecular characteristics of high-producing CHO clones, we developed a high throughput assay using RNAi and the Cell Xpress™ technology driven by LEAP (Laser Enabled Analysis and Processing) to evaluate the effects of targeted gene knockdown on therapeutic protein expression. Short interfering RNAs (siRNAs) were designed against genes associated with molecular pathways believed to be involved in the regulation of recombinant protein production and secretion. These siRNAs were transfected into recombinant CHO clones stably expressing humanized IgG. The transfected cells were then analyzed for cell growth, viability and productivity using Cell Xpress™. This approach uses the high throughput single cell imaging capabilities of the LEAP instrument to quantify recombinant protein secretion from individual cells. The same siRNAs were co-transfected into parental CHO cells along with the IgG light and heavy chain genes to confirm the results using a transient expression system. These experiments further our understanding of the mechanisms of CHO productivity.