Abstract
BackgroundEffective 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.ResultsWe 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.ConclusionWe 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.
Highlights
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 report the development of a versatile system which permits the knockdown of multiple target genes from a single transcript, and we show efficient depletion of endogenous pairs of signaling genes in the RAW264.7 murine macrophage-like cell line
To determine if multi-miR-shRNA transcripts could promote efficient and specific knockdown in mammalian cells, we first created plasmids expressing potent miR-shRNAs against the murine orthologs of three genes involved in G-protein signaling: arrestin 2 (Arr2), G-protein coupled receptor kinase 2 (Grk2) and G-protein beta 2 (Gβ2)
Summary
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. A variety of vector-based approaches, which express siRNAs as short hairpin (sh)RNAs, have been developed to permit delivery through viral vectors [3]. Designing such shRNA in the context of a naturally occurring RNA polymerase (pol) II-driven microRNA transcript (miRshRNA; Fig. 1a) increases the flexibility of this approach allowing for conditional and cell type-specific expression [4,5,6,7,8,9,10,11]. The observation that some endogenous microRNAs (miRNA) are processed from single transcripts containing multiple primary miRNA sequences [12,13] adds (page number not for citation purposes)
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