期刊
PLANT BIOTECHNOLOGY JOURNAL
卷 10, 期 8, 页码 936-944出版社
WILEY-BLACKWELL
DOI: 10.1111/j.1467-7652.2012.00722.x
关键词
BY-2; cell line optimization; fluorescence-activated cell sorting; monoclonal human antibody; Nicotiana tabacum; plant cell suspension culture; single-cell regeneration
资金
- Dow AgroSciences, Indianapolis
Plant cell suspension cultures can be used for the production of recombinant pharmaceutical proteins, but their potential is limited by modest production levels that may be unstable over long culture periods, reflecting initial culture heterogeneity and subsequent genetic and epigenetic changes. We used flow sorting to generate highly productive monoclonal cell lines from a heterogeneous population of tobacco BY-2 cells expressing the human antibody M12 by selecting the co-expressed fluorescent marker protein DsRed located on the same T-DNA. Separation yielded similar to 35% wells containing single protoplasts and similar to 15% wells with monoclonal microcolonies that formed within 2 weeks. Thus, enriching the population of fluorescent cells from initially 24% to 9096% in the six monoclonal lines resulted in an up to 13-fold increase in M12 production that remained stable for 1012 months. This is the first straightforward procedure allowing the generation of monoclonal plant cell suspension cultures by flow sorting, greatly increasing the potential of plant cells as an economical platform for the manufacture of recombinant pharmaceutical proteins.
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