Inducing RNAi in Drosophila cells by transfection with dsRNA.
Inducing RNAi in Drosophila cells by transfection with dsRNA.
复制标题
DOI：
10.1101/pdb.prot074351
复制
发表时间：
2013-05-01
影响因子：
--
通讯作者：
Perrimon N
中科院分区：
文献类型：
--
作者：
Zhou R;Mohr S;Hannon GJ;Perrimon N
在果蝇细胞中，合成的长双链RNA(DsRNAs)可以触发RNA干扰(RNAi)。对于许多果蝇细胞系和细胞类型来说，被动的dsRNA摄取是低效的。在果蝇S2细胞中，使用转基因技术通常可以获得更完整的沉默反应，这可能是因为细胞内dsRNA水平更高。在该方案中，使用QIAGEN的Effectene试剂将dsRNA导入S2细胞，这已被许多研究人员证明是可靠的。还可以将质粒DNA包括在转染组中以提供额外的功能。例如，质粒DNA可以编码一条途径或特定转录因子的活性报告，或允许对某些细胞行为或结构进行可视化的标记。包括编码荧光蛋白的质粒也是有用的，仅仅是为了监测转染率。
In Drosophila cells, RNA interference (RNAi) can be triggered by synthetic long double-stranded RNAs (dsRNAs). For many Drosophila cell lines and cell types, passive dsRNA uptake is inefficient. More complete silencing responses can often be obtained in Drosophila S2 cells using transfection, perhaps because higher levels of intracellular dsRNA are achieved. In this protocol, S2 cells are transfected with dsRNA using QIAGEN’s Effectene reagent, which has proven to be reliable for many investigators. A plasmid DNA can also be included in the transfection mix to provide additional functionality. The plasmid DNA can encode, for example, a reporter of the activity of a pathway or specific transcription factor, or a marker that allows visualization of some cellular behavior or structure. It is also useful to include a plasmid that encodes a fluorescent protein simply to monitor transfection efficiency.