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Open Access Highly Accessed Methodology article

A versatile approach to multiple gene RNA interference using microRNA-based short hairpin RNAs

Xiaocui Zhu, Leah A Santat, Mi Sook Chang, Jamie Liu, Joelle R Zavzavadjian, Estelle A Wall, Christine Kivork, Melvin I Simon and Iain DC Fraser*

Author Affiliations

The Alliance for Cellular Signaling, Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA

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BMC Molecular Biology 2007, 8:98  doi:10.1186/1471-2199-8-98

Published: 30 October 2007

Abstract

Background

Effective and stable knockdown of multiple gene targets by RNA interference is often necessary to overcome isoform redundancy, but it remains a technical challenge when working with intractable cell systems.

Results

We have developed a flexible platform using RNA polymerase II promoter-driven expression of microRNA-like short hairpin RNAs which permits robust depletion of multiple target genes from a single transcript. Recombination-based subcloning permits expression of multi-shRNA transcripts from a comprehensive range of plasmid or viral vectors. Retroviral delivery of transcripts targeting isoforms of cAMP-dependent protein kinase in the RAW264.7 murine macrophage cell line emphasizes the utility of this approach and provides insight to cAMP-dependent transcription.

Conclusion

We demonstrate functional consequences of depleting multiple endogenous target genes using miR-shRNAs, and highlight the versatility of the described vector platform for multiple target gene knockdown in mammalian cells.