(194e) Proteomics and Genome Sequencing to Enhance Chinese Hamster Ovary (CHO) Cell Productivity

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 protein spots 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. Here we review the effects of CD on SEAP producing cells in addition to observing the effects two other actin inhibitors, Cytochalasin E and Latrunculin B. Elongation factor 2, which aids translation, and PEST containing protein, associated with protein degradation, were also expressed at increased levels during amplification. We present our observations on the expression of these two proteins on SEAP productivity in CHO cells.

We also discuss the role of non-coding RNA (ncRNA) in CHO cells. Recent studies have shown that over 90% of transcripts in eukaryotic cells are not translated. These transcripts carry out a large variety of regulatory functions. We examine the usefulness of next generation short read sequencing technology as it relates to both ncRNA and genomic DNA in CHO cells. We present low coverage genomic Chinese hamster DNA sequence and its alignment to the mouse and rat genomes.