Article

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

The Alliance for Cellular Signaling, Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA.
BMC Molecular Biology (Impact Factor: 2.06). 02/2007; 8:98. DOI: 10.1186/1471-2199-8-98
Source: PubMed

ABSTRACT 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.
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.
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.

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