Comparison of Three Transposons for the Generation of Highly Productive Recombinant CHO Cell Pools and Cell Lines
Comparison of Three Transposons for the Generation of Highly Productive Recombinant CHO Cell Pools and Cell Lines
复制标题
DOI：
10.1002/bit.25888
复制
发表时间：
2016-06-01
影响因子：
3.8
通讯作者：
Wurm, Florian M.
中科院分区：
文献类型：
--
作者：
Balasubramanian, Sowmya;Rajendra, Yashas;Wurm, Florian M.
已经对几种天然存在的脊椎动物转座因子进行了遗传修饰，以使重组基因能够在哺乳动物细胞中转座。我们比较了三种转座子spiggyBac、Tol 2和Sleeping Beauty产生细胞池（重组细胞的多克隆培养物）和克隆细胞系的能力，以中国仓鼠卵巢细胞（CHO-DG 44）为宿主大规模生产重组蛋白。每个双载体转座子系统的转染导致在细胞池中的肿瘤坏死因子受体-Fc融合蛋白（TNFR：Fc）的体积产量约为9倍，高于那些从细胞池产生的常规质粒转染。平均而言，细胞池具有每个细胞10-12个整合的转基因拷贝。在没有选择的情况下，细胞池的体积生产率在2个月的培养期内下降了50%，然后保持恒定。从细胞池回收的克隆细胞系的平均体积TNFR：Fc生产率比通过常规转染产生的细胞系高约25倍。在14天的补料分批培养中，使用三种转座子中的任一种，从多克隆细胞池获得高达900 mg/L的TNFR：Fc水平，并且从克隆细胞系获得高达1.5g/L的TNFR：Fc水平。(C)2015 Wiley Periodicals，Inc.
Several naturally occurring vertebrate transposable elements have been genetically modified to enable the transposition of recombinant genes in mammalian cells. We compared three transposonspiggyBac, Tol2, and Sleeping Beautyfor their ability to generate cell pools (polyclonal cultures of recombinant cells) and clonal cell lines for the large-scale production of recombinant proteins using Chinese hamster ovary cells (CHO-DG44) as the host. Transfection with each of the dual-vector transposon systems resulted in cell pools with volumetric yields of tumor necrosis factor receptor-Fc fusion protein (TNFR:Fc) that were about ninefold higher than those from cell pools generated by conventional plasmid transfection. On average, the cell pools had 10-12 integrated copies of the transgene per cell. In the absence of selection, the volumetric productivity of the cell pools decreased by 50% over a 2-month cultivation period and then remained constant. The average volumetric TNFR:Fc productivity of clonal cell lines recovered from cell pools was about 25 times higher than that of cell lines generated by conventional transfection. In 14-day fed-batch cultures, TNFR:Fc levels up to 900mg/L were obtained from polyclonal cell pools and up to 1.5g/L from clonal cell lines using any of the three transposons. (C) 2015 Wiley Periodicals, Inc.