Proteomics Identifies Genetic Targets to Increase CHO Cell Productivity and the Use of Short Read Sequencing Technology for Studying Gene Regulation in CHO Cells

Methotrexate amplification was previously used to increase recombinant secreted alkaline phosphatase (SEAP) production in CHO cells. 2D gel electrophoresis and mass spectrometry were used to identify 21 proteins with consistently altered expression during amplification. One protein with increased expression, CapZ, inhibits growth of actin filaments. To mimic the effect of CapZ a small molecule drug and actin filament inhibitor, Cytochalasin D (CD), was used. CD was found to destabilize actin and increase SEAP productivity. The effects of CD and two other actin inhibiting drugs, Cytochalasin E and Latrunculin B, on SEAP producing cells and on cells producing tissue plasminogen activator (tPA) will be presented. High speed confocal microscopy is used to help visualize the effects CD has on cells with enhanced protein productivity. Simultaneous visualizations of the actin cytoskeleton, endoplasmic reticulum, cell membrane, and nuclei are presented in CD treated CHO cells. Data using next generation sequencing technology to better understand gene expression regulation will also be presented.