Vector-based RNA interference (RNAi) has emerged as a valuable tool for analysis of gene function. We have developed new RNA polymerase II expression vectors for RNAi, designated SIBR vectors, based upon the non-coding RNA BIC. BIC contains the miR-155 microRNA (miRNA) precursor, and we find that expression of a short region of the third exon of mouse BIC is sufficient to produce miR-155 in mammalian cells. The SIBR vectors use a modified miR-155 precursor stem-loop and flanking BIC sequences to express synthetic miRNAs complementary to target RNAs. Like RNA polymerase III driven short hairpin RNA vectors, the SIBR vectors efficiently reduce target mRNA and protein expression. The synthetic miRNAs can be expressed from an intron, allowing coexpression of a marker or other protein with the miRNAs. In addition, intronic expression of a synthetic miRNA from a two intron vector enhances RNAi. A SIBR vector can express two different miRNAs from a single transcript for effective inhibition of two different target mRNAs. Furthermore, at least eight tandem copies of a synthetic miRNA can be expressed in a polycistronic transcript to increase the inhibition of a target RNA. The SIBR vectors are flexible tools for a variety of RNAi applications.
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The authors thank Jenn-Yah Yu for helpful discussions, and Takahiko Matsuda and Connie Cepko for providing plasmids. The authors also thank Melissa Tippens for helping to set up small RNA northern blots, and Paul Good and David Engelke for the U6 probe and advice on northern blots. The authors gratefully acknowledge Lawrence Tsoi for assistance with constructing the multimerized SIBR luc vectors. This work was supported by NIMH MH073085 (A.B.V.), American Cancer Society (Research Scholar Grant RSG-01-177-01-MGO, A.B.V.), NIMH MH063992 (P.D.P.), NIH NS38698 (D.L.T.), the University of Michigan Center for Gene Therapy (D.L.T.), and the Wilson Medical Research Foundation (D.L.T.). Funding to pay the Open Access publication charges for this article was provided by the NIH and the University of Michigan.