Culture of Drosophila primary cells dissociated from gastrula embryos and their use in RNAi screening
Culture of Drosophila primary cells dissociated from gastrula embryos and their use in RNAi screening
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DOI：
10.1038/nprot.2009.147
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发表时间：
2009-01-01
期刊：
影响因子：
14.8
通讯作者：
Perrimon, Norbert
中科院分区：
文献类型：
--
作者：
Bai, Jianwu;Sepp, Katharine J.;Perrimon, Norbert
我们提供了一个详细的协议，大量培养的原代细胞从果蝇胚胎分离。该方案优于其他方案的优点是，我们已经对其进行了优化，以获得适合筛选的稳健的大规模性能。更重要的是，我们进一步提出了用双链（ds）RNA处理这些细胞以进行基因敲低的条件。果蝇原代细胞中的有效RNAi通过简单地将细胞浸泡在含有dsRNA的培养基中来实现。该方法为使用RNAi或小分子在分化细胞（如神经元和肌肉）中进行功能基因组筛选提供了基础。整个过程大约需要14 d，而果蝇胚胎原代细胞的制备仅需2-4 h。
We provide a detailed protocol for the mass culturing of primary cells dissociated from Drosophila embryos. The advantage of this protocol over others is that we have optimized it for a robust large-scale performance that is suitable for screening. More importantly, we further present conditions to treat these cells with double stranded (ds) RNAs for gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. This method provides the basis for functional genomic screens in differentiated cells, such as neurons and muscles, using RNAi or small molecules. The entire protocol takes similar to 14 d, whereas the preparation of primary cells from Drosophila embryos only requires 2-4 h.