中国仓鼠卵巢细胞
转染
生物
转基因
细胞培养
分子生物学
细胞生物学
重组DNA
转座酶
转座因子
基因
遗传学
突变体
作者
Mattia Matasci,Lucia Baldi,David L. Hacker,Florian Μ. Wurm
摘要
Abstract Generating stable, high‐producing mammalian cell lines is a major bottleneck in the manufacture of recombinant therapeutic proteins. Conventional gene transfer methods for cell line generation rely on random plasmid integration, resulting in unpredictable and highly variable levels of transgene expression. As a consequence, a large number of stably transfected cells must be analyzed to recover a few high‐producing clones. Here we present an alternative gene transfer method for cell line generation based on transgene integration mediated by the piggyBac (PB) transposon. Recombinant Chinese hamster ovary (CHO) cell lines expressing a tumor necrosis factor receptor:Fc fusion protein were generated either by PB transposition or by conventional transfection. Polyclonal populations and isolated clonal cell lines were characterized for the level and stability of transgene expression for up to 3 months in serum‐free suspension culture. Pools of transposed cells produced up to fourfold more recombinant protein than did the pools generated by standard transfection. For clonal cell lines, the frequency of high‐producers was greater following transposition as compared to standard transfection, and these clones had a higher volumetric productivity and a greater number of integrated transgenes than did those generated by standard transfection. In general, the volumetric productivity of the cell pools and individual cell lines generated by transposition was stable for up to 3 months in the absence of selection. Our results indicate that the PB transposon supports the generation of cell lines with high and stable transgene expression at an elevated frequency relative to conventional transfection. Thus, PB‐mediated gene delivery is expected to reduce the extent of recombinant cell line screening. Biotechnol. Bioeng. 2011;108:2141–2150. © 2011 Wiley Periodicals, Inc.
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