Influence of genetic factors on the productivity of recombinant Chinese hamster ovary (CHO) cells.

摘要

In this work, influence of some genetic factors on the productivity of recombinant Chinese hamster ovary (CHO) cells has been studied using two systems. In the first system, we have investigated the effect of cloned gene dosage on cell growth and Hepatitis B surface antigen (HbsAg) synthesis and secretion in recombinant CHO cells. Gene copy numbers in each clone determined using Southern hybridizations were positively correlated with intracellular dihydrofolate reductase (DHFR) content using a flow cytometric assay. The mRNA levels determined using Northern hybridization also gave the same trends. Flow cytometry experiments also show that the amplified clones exhibit a great deal of heterogeneity in single-cell DHFR content as compared to parental cells. Specific growth rates in batch culture were found decrease with gene copy number. Secreted HbsAg titers and specific HbsAg secretion rates were found to increase with gene copy number.;Pulse-chase experiments show that efficiency of HbsAg secretion (fraction of total initial HbsAg (0 h chase) that is secreted into the extracellular medium during a 23.5 h chase) decreases and overall efficiency of HbsAg expression (relative amount of HbsAg secreted into the medium, (AU.mm/10;The second system we have used is a CHO cell line producing secreted tissue plasminogen activator (tPA). ATP limitations may influence productivity of secreted cloned proteins in CHO cells and expression of Vitreoscilla hemoglobin (VHb) has been suggested to increase ATP production efficiency in Escherichia coli. Based on these hypotheses, we have cloned the Vitreoscilla hemoglobin gene in CHO cells producing tPA (CHO-tPA) and obtained inducible intracellular expression of VHb in these transfected cells (VHb-CHO). Batch culture experiments show that VHb-CHO cells have a lower specific growth rate compared to CHO-tPA cells but these cells exhibit a significantly higher specific tPA production rate throughout the batch.