The integration of a major hepatitis B virus gene into cell-cycle synchronized carrot cell suspension cultures and its expression in regenerated carrot plants

The percentage of cells successfully transformed by Agrobacteria is usually very low (not more than 10% and usually much less; Thomas et al., 1989). In this report we show that in carrot (Daucus carota L., ssp. sativus) cell suspensions transformation efficiency was strongly improved by using cell cycle synchronized cells. Fluorodesoxyuridine (FDU) was added for 24 h to inhibit thymidine synthesis. This blocked the cell cycle at the transition from G1- to S-phase. Then the block was released by applying thymidine. A high rate of transformation was obtained when Agrobacterium tumefaciens was added concurrently with thymidine. As examples of efficient and long term foreign gene expression in transgenic cells, the reporter enzyme -glucuronidase (GUS) as a model as well as the major hepatitis B virus surface protein were used. Both genes were linked to the MAS promoter. In carrot cell suspensions containing the viral gene, the corresponding viral protein was produced. In roots of mature transgenic carrot plants generated through somatic embryogenesis and raised in soil as well as in callus cultures derived thereof, the viral protein was also produced.     

Virus susceptibility of Chinese hamster ovary (CHO) cells and detection of viral contaminations by adventitious agent testing

Biopharmaceuticals are of increasing importance in the treatment of a variety of diseases. A remaining concern associated with their production is the potential introduction of adventitious agents into their manufacturing process, which may compromise the pathogen safety of a product and potentially cause stock‐out situations for important medical supplies. To ensure the safety of biological therapeutics, regulatory guidance requires adventitious agent testing (AAT) of the bulk harvest. AAT is a deliberately promiscuous assay procedure which has been developed to indicate, ideally, the presence of any viral contaminant. One of the most important cell lines used in the production of biopharmaceuticals is Chinese hamster ovary (CHO) cells and while viral infections of CHO cells have occurred, a systematic screen of their virus susceptibility has never been published. We investigated the susceptibility of CHO cells to infection by 14 different viruses, including members of 12 families and representatives or the very species that were implicated in previously reported production cell infections. Based on our results, four different infection outcomes were distinguished, based on the possible combinations of the two factors (i) the induction, or not, of a cytopathic effect and (ii) the ability, or not, to replicate in CHO cells. Our results demonstrate that the current AAT is effective for the detection of viruses which are able to replicate in CHO cells. Due to the restricted virus susceptibility of CHO cells and the routine AAT of bulk harvests, our results provide re‐assurance for the very high safety margins of CHO cell‐derived biopharmaceuticals. Biotechnol. Bioeng. 2010;106: 598–607. © 2010 Wiley Periodicals, Inc.