CSIRO Livestock Industries, Australian Animal Health Laboratory, Geelong 3220, Australia.
Biochem Biophys Res Commun. 2011 Dec 9;416(1-2):194-8. doi: 10.1016/j.bbrc.2011.11.024. Epub 2011 Nov 10.
The U6 and 7SK RNA polymerase III promoters are widely used in RNAi research for the expression of shRNAs. However, with their increasing use in vitro and in vivo, issues associated with cytotoxicity have become apparent with their use. Therefore, alternative promoters such as the weaker H1 promoter are becoming a popular choice. With interest in the chicken as a model organism, we aimed to identify and characterise the chicken H1 promoter for the expression of shRNAs for the purpose of RNAi. The chicken H1 promoter was isolated and sequence analysis identified conserved RNA polymerase III promoter elements. A shRNA expression cassette containing the chicken H1 promoter and shRNA targeting enhanced green fluorescent protein (EGFP) was developed. An RNAse protection assay confirmed activity of the promoter determined by the detection of expressed shRNAs. Comparison of the H1 promoter to the chicken RNA polymerase III 7SK and U6 promoters demonstrated that expressed shRNAs from the H1 promoter induced gene specific silencing, albeit to lower levels in comparison to both 7SK and U6 promoters. Here we have identified a new tool for RNAi research with specific applications to the chicken. The availability of a RNA polymerase III promoter that drives shRNA expression to reduced levels will greatly benefit in ovo/in vivo applications where there are concerns of cytotoxicity resulting from overexpression of an shRNA.
U6 和 7SK RNA 聚合酶 III 启动子广泛用于 shRNA 的表达的 RNAi 研究。然而，随着它们在体外和体内的使用越来越多，与细胞毒性相关的问题随着它们的使用而变得明显。因此，像较弱的 H1 启动子这样的替代启动子成为了一个受欢迎的选择。由于对鸡作为模型生物的兴趣，我们旨在鉴定和表征鸡 H1 启动子，以表达用于 RNAi 的 shRNA。分离了鸡 H1 启动子，序列分析鉴定了保守的 RNA 聚合酶 III 启动子元件。开发了一个包含鸡 H1 启动子和靶向增强型绿色荧光蛋白 (EGFP) 的 shRNA 表达盒。RNA 酶保护测定法证实了启动子的活性，通过检测表达的 shRNA 来确定。将 H1 启动子与鸡 RNA 聚合酶 III 7SK 和 U6 启动子进行比较表明，来自 H1 启动子的表达 shRNA 诱导基因特异性沉默，尽管与 7SK 和 U6 启动子相比，沉默水平较低。在这里，我们已经为 RNAi 研究确定了一种新工具，特别适用于鸡。具有降低水平驱动 shRNA 表达的 RNA 聚合酶 III 启动子的可用性将极大地有益于胚胎内/体内应用，因为担心过量表达 shRNA 会导致细胞毒性。
