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

Zhu X, Santat LA, Chang MS, Liu J, Zavzavadjian JR, Wall EA, Kivork C, Simon MI, Fraser ID - BMC Mol. Biol. (2007)

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Alliance for Cellular Signaling, Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA. xiaocui_zhu@hotmail.com

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.

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Expression of concatenated miR30-based shRNAs in a single transcript can promote efficient knockdown of at least three target genes. (a) Predicted secondary structure of the unprocessed miR30 transcript. Arrow depicts the location of the mature miRNA which is replaced with gene specific siRNA sequence in the expressed miR-shRNA. (b) Proposed structure of a transcript expressing multiple gene specific miR-shRNAs. (c) Subcloning scheme for creation of multi-miR-shRNA transcripts. Prom; any of the pol II promoters listed in Fig. 2a, attL1 + attL2; Gateway recombination sites, 5'miR + 3'miR; flanking sequence derived from human miR30. (d) Single CMV promoter-driven miR-shRNAs against arrestin 2 (Arr2), G beta 2 (Gβ2) and G-protein coupled receptor kinase 2 (Grk2) promote potent and specific knockdown when co-expressed with YFP-tagged cDNAs of their respective target genes in HEK293 cells. (e) Single transcripts expressing different combinations of double miR-shRNAs promote efficient dual target knockdown. (f) A single transcript expressing miR-shRNA against Arr2, Gβ2 and Grk2 retains potency against all three target genes.
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Figure 1: Expression of concatenated miR30-based shRNAs in a single transcript can promote efficient knockdown of at least three target genes. (a) Predicted secondary structure of the unprocessed miR30 transcript. Arrow depicts the location of the mature miRNA which is replaced with gene specific siRNA sequence in the expressed miR-shRNA. (b) Proposed structure of a transcript expressing multiple gene specific miR-shRNAs. (c) Subcloning scheme for creation of multi-miR-shRNA transcripts. Prom; any of the pol II promoters listed in Fig. 2a, attL1 + attL2; Gateway recombination sites, 5'miR + 3'miR; flanking sequence derived from human miR30. (d) Single CMV promoter-driven miR-shRNAs against arrestin 2 (Arr2), G beta 2 (Gβ2) and G-protein coupled receptor kinase 2 (Grk2) promote potent and specific knockdown when co-expressed with YFP-tagged cDNAs of their respective target genes in HEK293 cells. (e) Single transcripts expressing different combinations of double miR-shRNAs promote efficient dual target knockdown. (f) A single transcript expressing miR-shRNA against Arr2, Gβ2 and Grk2 retains potency against all three target genes.

Mentions: 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 (miR-shRNA; Fig. 1a) increases the flexibility of this approach allowing for conditional and cell type-specific expression [4-11]. The observation that some endogenous microRNAs (miRNA) are processed from single transcripts containing multiple primary miRNA sequences [12,13] adds to the potential experimental utility of this approach to RNAi. In using RNAi as an experimental tool, the ability to effectively deplete multiple gene targets is vital to address the issue of isoform redundancy, especially in mammalian cell systems. Thus, a flexible vector platform that allows for effective multi-gene knockdown in essentially any cell system would be of great value.


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

Zhu X, Santat LA, Chang MS, Liu J, Zavzavadjian JR, Wall EA, Kivork C, Simon MI, Fraser ID - BMC Mol. Biol. (2007)

Expression of concatenated miR30-based shRNAs in a single transcript can promote efficient knockdown of at least three target genes. (a) Predicted secondary structure of the unprocessed miR30 transcript. Arrow depicts the location of the mature miRNA which is replaced with gene specific siRNA sequence in the expressed miR-shRNA. (b) Proposed structure of a transcript expressing multiple gene specific miR-shRNAs. (c) Subcloning scheme for creation of multi-miR-shRNA transcripts. Prom; any of the pol II promoters listed in Fig. 2a, attL1 + attL2; Gateway recombination sites, 5'miR + 3'miR; flanking sequence derived from human miR30. (d) Single CMV promoter-driven miR-shRNAs against arrestin 2 (Arr2), G beta 2 (Gβ2) and G-protein coupled receptor kinase 2 (Grk2) promote potent and specific knockdown when co-expressed with YFP-tagged cDNAs of their respective target genes in HEK293 cells. (e) Single transcripts expressing different combinations of double miR-shRNAs promote efficient dual target knockdown. (f) A single transcript expressing miR-shRNA against Arr2, Gβ2 and Grk2 retains potency against all three target genes.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2194719&req=5

Figure 1: Expression of concatenated miR30-based shRNAs in a single transcript can promote efficient knockdown of at least three target genes. (a) Predicted secondary structure of the unprocessed miR30 transcript. Arrow depicts the location of the mature miRNA which is replaced with gene specific siRNA sequence in the expressed miR-shRNA. (b) Proposed structure of a transcript expressing multiple gene specific miR-shRNAs. (c) Subcloning scheme for creation of multi-miR-shRNA transcripts. Prom; any of the pol II promoters listed in Fig. 2a, attL1 + attL2; Gateway recombination sites, 5'miR + 3'miR; flanking sequence derived from human miR30. (d) Single CMV promoter-driven miR-shRNAs against arrestin 2 (Arr2), G beta 2 (Gβ2) and G-protein coupled receptor kinase 2 (Grk2) promote potent and specific knockdown when co-expressed with YFP-tagged cDNAs of their respective target genes in HEK293 cells. (e) Single transcripts expressing different combinations of double miR-shRNAs promote efficient dual target knockdown. (f) A single transcript expressing miR-shRNA against Arr2, Gβ2 and Grk2 retains potency against all three target genes.
Mentions: 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 (miR-shRNA; Fig. 1a) increases the flexibility of this approach allowing for conditional and cell type-specific expression [4-11]. The observation that some endogenous microRNAs (miRNA) are processed from single transcripts containing multiple primary miRNA sequences [12,13] adds to the potential experimental utility of this approach to RNAi. In using RNAi as an experimental tool, the ability to effectively deplete multiple gene targets is vital to address the issue of isoform redundancy, especially in mammalian cell systems. Thus, a flexible vector platform that allows for effective multi-gene knockdown in essentially any cell system would be of great value.

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Alliance for Cellular Signaling, Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA. xiaocui_zhu@hotmail.com

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.

Show MeSH
Related in: MedlinePlus