Journal of RNAi and Gene Silencing (2007), 3(1), 237-247

Reseacrh Article

A novel expression system for artificial miRNAs containing no endogenous miRNA precursor sequences

Atsushi Shibata *, Akiko Iwaki and Yasuyuki Fukumaki

ABSTRACT

Recently, artificial microRNA (miRNA)-mediated RNA interference (RNAi) systems have been developed as useful tools to study gene functions. We report an artificial miRNA precursor motif (AMPM) containing several restriction sites in the loop and in the extended stem structures, and generated a vector-based expression system for AMPM under the control of an RNA polymerase II promoter. The AMPM located in the intron or the exon of the selection marker gene mediated silencing of the reporter luciferase gene expression in HeLa cells. Northern blotting and colony formation assays revealed that the AMPM was efficiently and appropriately processed into mature miRNA. The AMPA system also downregulated endogenous p53 and laminA/C proteins in stable transfectants. Moreover, clustered AMPMs separated by an appropriate spacer and targeting different sites within a single mRNA mediated gene silencing even if the sequences of the target and the AMPM were partially complementary, suggesting the potential therapeutic utility of clustered AMPMs for highly mutable targets, such as human immunodeficiency virus type 1.

KEYWORDS: microRNA, miRNA, cluster, RNA polymerase II, HIV-1, gene therapy

Division of Disease Genes, Research Center for Genetic Information, Medical Institute of Bioregulation, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan

*Correspondence to: Atsushi Shibata, Email: ash@gen.kyushu-u.ac.jp, Tel: +81 92 642 6168, Fax: +81 92 632 2375

© Copyright Atsushi Shibata et al

(Received ; Revised ; Accepted )