TRANSFECTION OF MAMMALIAN-CELLS WITH PLASMID DNA BY SCRAPE LOADING AND SONICATION LOADING
TRANSFECTION OF MAMMALIAN-CELLS WITH PLASMID DNA BY SCRAPE LOADING AND SONICATION LOADING
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DOI：
10.1073/pnas.84.23.8463
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发表时间：
1987-12-01
影响因子：
11.1
通讯作者：
ZIMMER, SG
中科院分区：
文献类型：
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作者：
FECHHEIMER, M;BOYLAN, JF;ZIMMER, SG
刮片加载和超声加载是两种最近描述的将大分子引入活细胞的方法。我们已经测试了这些方法用外源DNA转染哺乳动物细胞的功效，使用基于对药物G418（遗传霉素）的抗性或基于获得利用嘧啶生物合成的补救途径的能力的选择系统。这些加载方法可用于产生瞬时和稳定表达转染的DNA分子的基因产物的细胞系。当将DNA加入到缺乏二价阳离子并含有K+代替Na+的生理盐水中的细胞中时，观察到最佳转染。DNA分子7.1至30个碱基长已通过刮涂加载程序引入。此外，刮取加载程序已被用于共转染和随后表达在与其表达被选择的DNA分子的混合物中添加的DNA分子上编码的非选择性基因。通过这种方法已经获得了表达对调节细胞生长和分裂重要的癌基因或蛋白质的细胞系。刮片加载程序也可用于研究外源基因表达后发生的细胞变化。在转染后1 - 5天，多达80%的细胞刮擦装载有编码猿猴病毒40大肿瘤抗原的质粒pC6，在细胞核中含有这种蛋白质。因此，刮加载和超声加载是简单的，经济的，可重复的方法，用于将DNA分子引入粘附和非粘附细胞，这些方法可能是有用的，在未来的实验在基础和应用水平。
Scrape loading and sonication loading are two recently described methods of introducing macromolecules into living cells. We have tested the efficacy of these methods for transfection of mammalian cells with exogenous DNA, using selection systems based either on resistance to the drug G418 (Geneticin) or on acquisition of the ability to ulilize the salvage pathway of pyrimidine biosynthesis. These loading methods can be employed to generate cell lines that express the gene product of the transfected DNA molecules both transiently and stably. Optimal transfection is observed when the DNA is added to cells in physiological saline lacking divalent cations and containing K+ in place of Na+. DNA molecules 7.1 to 30 kilobases long have been introduced by the scrape loading procedure. In addition, the scrape loading procedure has been employed for cotransfection and subsequent expression of nonselectable genes encoded on DNA molecules added in a mixture with DNA molecules whose expression is selected. Cell lines expressing oncogenes or proteins that are important for regulation of cell growth and division have been obtained by this procedure. The scrape loading procedure is also useful for studies of the cellular changes that occur upon expression of an exogenous gene. As many as 80% of cells scrape loaded with the plasmid pC6, which encodes the simian virus 40 large tumor antigen, contained this protein in the nucleus between 1 and 5 days after transfection. Thus, scrape loading and sonication loading are simple, economical, and reproducible methods for introduction of DNA molecules into adherent and nonadherent cells, and these methods may be useful in the future for experimentation at both fundamental and applied levels.