Liposome-mediated transfection.
Liposome-mediated transfection.
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DOI：
10.1101/pdb.prot5583
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发表时间：
2011-03
影响因子：
--
通讯作者：
D. Dean;J. Gasiorowski
中科院分区：
文献类型：
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作者：
D. Dean;J. Gasiorowski
简介 将外源 DNA 有效递送至细胞进行表达和功能研究是现代细胞生物学的一项基本技术。阳离子脂质体是最广泛使用的转染载体，有多种市售产品：Lipofectin（DOTMA [N-[1(2,3-二油酰氧基)丙基]-N,N,N-三甲基氯化铵]和 DOPE [二油酰磷脂酰乙醇胺] 的 1:1 混合物）、Transfectam 和 DOTAP [1,2-二油酰氧基-3-三甲基丙烷铵]：DOPE。然而，所有这些试剂都很昂贵；购买纯化的脂质并通过添加等摩尔量的悬浮在氯仿中的每种脂质、混合并在惰性气体下干燥来配制自制转染试剂可以更便宜。试剂可以保存直至在适当的缓冲液中重新配制。尽管每个制造商都提供了其试剂使用的详细说明，但以下一般步骤基本上适用于所有配方；时间、浓度和体积可能略有不同。以下体积足以转染 35 毫米培养皿或六孔培养皿中的一个孔。
INTRODUCTIONThe efficient delivery of exogenous DNA to cells for expression and function studies is an essential technique of modern cell biology. Cationic liposomes are the most widely used class of carrier for transfection and a number of products are available commercially: Lipofectin (a 1:1 mixture of DOTMA [N-[1(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride] and DOPE [dioleoyl phosphatidylethanolamine]), Transfectam, and DOTAP [1,2-dioleoyloxy-3-trimethylammonium propane]:DOPE. However, all these reagents are costly; it can be less expensive to purchase purified lipids and formulate homemade transfection reagents by adding equimolar amounts of each lipid suspended in chloroform, mixing, and drying under inert gas. The reagents can be stored until they are reconstituted in an appropriate buffer. Although each manufacturer provides detailed instructions for the use of its reagent, the following general steps are used for essentially all formulations; times, concentrations, and volumes might differ slightly. The following volumes are sufficient for transfecting a 35-mm dish or one well of a six-well dish.