Title

A novel short hairpin RNA (shRNA) expression system promotes Sox9-dependent gene silencing

Document Type

Article

Publication Date

2009

Abstract

Cartilage development and function are dependent on a temporally integrated program of gene expression. With the advent of RNA interference (RNAi), artificial control of these complex programs becomes a possibility, limited only by the ability to regulate and express the RNAi. Using existing methods for production of RNAi's, we have constructed a plasmid-based short hairpin RNA (shRNA) expression system under control of the human pol III H1 promoter and supplemented this promoter with DNA binding sites for the cartilage-specific transcription factor Sox9. The resulting shRNA expression system displays robust, Sox9-dependent gene silencing. Dependence on Sox9 expression was confirmed by electrophoretic mobility shift assays. The ability of the system to regulate heterologously expressed Sox9 was demonstrated by Western blot, as a function of both Sox9 to shRNA ratio, as well as time from transfection. This novel expression system supports auto-regulatory gene silencing, providing a tissue-specific feedback mechanism for temporal control of gene expression. Its applications for both basic mechanistic studies and therapeutic purposes should facilitate the design and implementation of innovative tissue engineering strategies. © 2009 Elsevier Inc. All rights reserved.

Publication Title

Plasmid

Volume

62

Issue

1

First Page

50

Last Page

55

Comments

This article was published in Plasmid, Volume 62, Issue 1, Pages 50-55.

The published version is available at http://dx.doi.org/10.1016/j.plasmid.2009.04.001.

Copyright © 2009.

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