Transient mammalian cell transfection with polyethylenimine (PEI).
Transient mammalian cell transfection with polyethylenimine (PEI).
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DOI：
10.1016/b978-0-12-418687-3.00018-5
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发表时间：
2013
影响因子：
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通讯作者：
Leahy, Daniel J.
中科院分区：
文献类型：
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作者：
Longo, Patti A.;Kavran, Jennifer M.;Kim, Min-Sung;Leahy, Daniel J.
标准蛋白表达系统，如E.大肠杆菌，通常不能产生折叠的、单分散的或功能性的真核蛋白质（参见大肠杆菌中蛋白质的小规模表达。大肠杆菌）。使用含有适当折叠和翻译后机制的真核系统（例如哺乳动物细胞）对这些蛋白质的表达大有裨益。在这里，我们描述了使用聚乙烯亚胺（PEI）在哺乳动物细胞中进行小规模和大规模瞬时表达的方法。我们发现这种方法比传统的产生稳定细胞系的方法更经济有效，更快。首先，使用贴壁细胞在小规模上确定最佳转染条件。然后将这些条件转化用于大规模悬浮培养。有关生成稳定细胞系的更多详细信息，请参见使用Gateway®纯化克隆技术和Flp-In T-REx®系快速创建稳定的哺乳动物细胞系以调节蛋白质表达或通过电穿孔生成哺乳动物稳定细胞系）。
Standard protein expression systems, such as E. coli, often fail to produce folded, mono-disperse, or functional eukaryotic proteins (see Small-scale Expression of Proteins in E. coli). The expression of these proteins is greatly benefited by using a eukaryotic system, such as mammalian cells, that contains the appropriate folding and posttranslational machinery. Here, we describe methods for both small- and large-scale transient expression in mammalian cells using polyethylenimine (PEI). We find this procedure to be more cost-effective and quicker than the more traditional route of generating stable cell lines. First, optimal transfection conditions are determined on a small-scale, using adherent cells. These conditions are then translated for use in large-scale suspension cultures. For further details on generating stable cell lines please (see Rapid creation of stable mammalian cell lines for regulated expression of proteins using the Gateway® Recombination Cloning Technology and Flp-In T-REx® lines or Generating mammalian stable cell lines by electroporation).