RNA interference rescue by bacterial artificial chromosome transgenesis in mammalian tissue culture cells
RNA interference rescue by bacterial artificial chromosome transgenesis in mammalian tissue culture cells
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DOI：
10.1073/pnas.0409861102
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发表时间：
2005-02-15
影响因子：
11.1
通讯作者：
Buchholz, F
中科院分区：
文献类型：
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作者：
Kittler, R;Pelletier, L;Buchholz, F
RNA干扰（RNAi）是一种广泛应用于组织培养细胞中基因功能分析的方法。然而，迄今为止，还没有可靠的方法来测试任何特定RNAi实验的特异性。理想的实验是通过以RNAi难治的形式表达靶基因来拯救表型。转基因应在生理水平表达，并具有其不同的剪接变体。在这里，我们证明了鼠细菌人工染色体在人类细胞中的表达提供了一种可靠的方法来创建RNAi抗性转基因。这种策略应该适用于所有真核生物，因此应该成为确认RNAi特异性的标准技术。我们表明，这种技术可以扩展到允许创建标记的转基因，在生理水平上表达，基因功能的进一步研究。
RNA interference (RNAi) is a widely used method for analysis of gene function in tissue culture cells. However, to date there has been no reliable method for testing the specificity of any particular RNAi experiment. The ideal experiment is to rescue the phenotype by expression of the target gene in a form refractory to RNAi. The transgene should be expressed at physiological levels and with its different splice variants. Here, we demonstrate that expression of murine bacterial artificial chromosomes in human cells provides a reliable method to create RNAi-resistant transgenes. This strategy should be applicable to all eukaryotes and should therefore be a standard technology for confirming the specificity of RNAi. We show that this technique can be extended to allow the creation of tagged transgenes, expressed at physiological levels, for the further study of gene function.