来自RNA聚合酶III启动子的干扰RNA的多顺反子表达。

Polycistronic expression of interfering RNAs from RNA polymerase III promoters.

作者信息

Steel Laura F, Sanghvi Viraj R

机构信息

Department of Microbiology and Immunology, Institute for Molecular Medicine and Infectious Disease, Drexel University College of Medicine, 245 North 15th Street, Philadelphia, PA 19102, USA.

出版信息

Methods Mol Biol. 2012;815:347-59. doi: 10.1007/978-1-61779-424-7_26.

DOI:10.1007/978-1-61779-424-7_26

PMID:22131004

Abstract

In many RNA silencing applications, there is a benefit to expressing multiple interfering RNAs simultaneously. This can be achieved by using a single RNA polymerase II promoter to express multiple micro(mi)RNA-formatted interfering RNAs that are arranged in a polycistronic cluster, mimicking the organization of naturally clustered, endogenous miRNAs. While RNA pol III promoters are often used to express individual short hairpin (sh) RNAs, we have recently shown that pol III promoters can also be used to drive polycistronic expression of miRNA-formatted interfering RNAs. Here, we present methods for the assembly of polycistronic miRNA expression vectors that use pol III promoters. In addition, we present methods for testing the potency and the level of expression of each of the individual miRNAs encoded in the construct.

摘要

在许多RNA沉默应用中，同时表达多种干扰RNA是有益的。这可以通过使用单个RNA聚合酶II启动子来表达多个以多顺反子簇形式排列的微小（mi）RNA格式干扰RNA来实现，模仿天然成簇的内源性miRNA的组织形式。虽然RNA聚合酶III启动子常用于表达单个短发夹（sh）RNA，但我们最近表明，聚合酶III启动子也可用于驱动miRNA格式干扰RNA的多顺反子表达。在此，我们介绍了使用聚合酶III启动子组装多顺反子miRNA表达载体的方法。此外，我们还介绍了测试构建体中编码的每个单个miRNA的效力和表达水平的方法。