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Published October 30, 2007 | Published
Journal Article Open

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


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.

Additional Information

© 2007 Zhu et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Received: 1 June 2007. Accepted: 30 October 2007. Published: 30 October 2007. We are grateful to colleagues in the AfCS for discussions and technical advice and to Lucas Cheadle for technical assistance. We thank Linda Holloway, Jason Cooper and Unice Na at the ATCC for assistance in making the plasmids described in this study available to the research community. This work was supported by contributions from public and private sources, including the NIGMS Glue Grant Initiative (U54 GM062114). A complete listing of the Alliance for Cellular Signaling (AfCS) sponsors can be found on the AfCS website [29].

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