A robust system for RNA interference in the chicken using a modified microRNA operon
A robust system for RNA interference in the chicken using a modified microRNA operon
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DOI：
10.1016/j.ydbio.2006.02.020
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发表时间：
2006-06-15
影响因子：
2.7
通讯作者：
Wilson, Stuart A.
中科院分区：
文献类型：
--
作者：
Das, Raman M.;Van Hateren, Nick J.;Wilson, Stuart A.
RNA干扰（RNA interference，RNAi）技术为沉默基因表达和研究基因功能提供了一种有效的方法。然而，用于鸡胚胎的RNAi工具在很大程度上改编自为哺乳动物细胞设计的载体。在这里，我们提出了质粒和逆转录病毒RNAi载体，专门设计用于鸡细胞中的最佳基因沉默。这些载体使用鸡U6启动子来表达以microRNA30为模型的RNA，这些RNA嵌入鸡microRNA操纵子序列中，以确保转录物的最佳Drosha和Dicer加工。鸡U6启动子的作用明显优于哺乳动物来源的启动子，并且与microRNA操纵子表达盒（MOEC）组合，实现了高达90%的靶基因沉默。通过使用MOEC，我们表明，它也可以同时沉默两个基因与一个单一的载体。载体表达REP或GFP标记，允许载体递送的简单体内追踪。使用这些质粒，我们证明了有效的沉默Pax3，Pax6，Nkx2.1，Nkx2.2，Notch1和Shh在鸡胚胎神经系统的离散区域。该RNAi系统的高效性和易用性为鸡胚胎中的大规模遗传筛选铺平了道路。(c)2006年爱思唯尔公司All rights reserved.
RNA interference (RNAi) provides an effective method to silence gene expression and investigate gene function. However, RNAi tools for the chicken embryo have largely been adapted from vectors designed for mammalian cells. Here we present plasmid and retroviral RNAi vectors specifically designed for optimal gene silencing in chicken cells. The vectors use a chicken U6 promoter to express RNAs modelled on microRNA30, which are embedded within chicken microRNA operon sequences to ensure optimal Drosha and Dicer processing of transcripts. The chicken U6 promoter works significantly better than promoters of mammalian origin and in combination with a microRNA operon expression cassette (MOEC), achieves up to 90% silencing of target genes. By using a MOEC, we show that it is also possible to simultaneously silence two genes with a single vector. The vectors express either REP or GFP markers, allowing simple in vivo tracking of vector delivery. Using these plasmids, we demonstrate effective silencing of Pax3, Pax6, Nkx2.1, Nkx2.2, Notch1 and Shh in discrete regions of the chicken embryonic nervous system. The efficiency and ease of use of this RNAi system paves the way for large-scale genetic screens in the chicken embryo. (c) 2006 Elsevier Inc. All rights reserved.