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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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miR-shRNA expression options. A) Flow chart for expressing miR-shRNA in different expression platforms. Experimental details of the various steps are provided in methods. B) Comparison of MSCV LTR and beta actin promoters expressing arrestin 2 (Arr2) miR-shRNA or control luciferase (Luc) miR-shRNA in lentivirally infected RAW264.7 macrophages. Constructs used for lentivirus production are detailed in Additional file 3.
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Figure 5: miR-shRNA expression options. A) Flow chart for expressing miR-shRNA in different expression platforms. Experimental details of the various steps are provided in methods. B) Comparison of MSCV LTR and beta actin promoters expressing arrestin 2 (Arr2) miR-shRNA or control luciferase (Luc) miR-shRNA in lentivirally infected RAW264.7 macrophages. Constructs used for lentivirus production are detailed in Additional file 3.

Mentions: We engineered the vectors to be extremely versatile with a high degree of flexibility for downstream applications (Fig. 5a and Additional file 1). We created the initial shRNA cloning/testing entry vector with a choice of up to five interchangeable promoters (CMV, EF1, beta-actin, ubiquitin-c and MSCV 5'LTR), which can be combined with six different selection markers upon recombination to a selection of lentiviral vectors (Fig. 5a). This allows selection of the optimal promoter for a given cell system; for example, comparison of MSCV LTR and β-actin promoters driving the Arr2 miR-shRNA in RAW cells shows the former promotes more potent knockdown (Fig. 5b). Alternatively, the entry vector promoter can be removed after shRNA validation for recombination to a variety of expression platforms with existing promoters. These include 5'LTR-driven retroviral vectors or CMV/EF1-driven mammalian expression vectors which can then be used for creation of stable cell lines. These latter vectors also contain a broad selection of co-expressed fluorescent and/or drug selection markers (Additional file 1). Furthermore, the Gateway compatibility of these vectors allows the miR-shRNA cassettes to be recombined to any appropriately configured Gateway-ready expression system.


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)

miR-shRNA expression options. A) Flow chart for expressing miR-shRNA in different expression platforms. Experimental details of the various steps are provided in methods. B) Comparison of MSCV LTR and beta actin promoters expressing arrestin 2 (Arr2) miR-shRNA or control luciferase (Luc) miR-shRNA in lentivirally infected RAW264.7 macrophages. Constructs used for lentivirus production are detailed in Additional file 3.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: miR-shRNA expression options. A) Flow chart for expressing miR-shRNA in different expression platforms. Experimental details of the various steps are provided in methods. B) Comparison of MSCV LTR and beta actin promoters expressing arrestin 2 (Arr2) miR-shRNA or control luciferase (Luc) miR-shRNA in lentivirally infected RAW264.7 macrophages. Constructs used for lentivirus production are detailed in Additional file 3.
Mentions: We engineered the vectors to be extremely versatile with a high degree of flexibility for downstream applications (Fig. 5a and Additional file 1). We created the initial shRNA cloning/testing entry vector with a choice of up to five interchangeable promoters (CMV, EF1, beta-actin, ubiquitin-c and MSCV 5'LTR), which can be combined with six different selection markers upon recombination to a selection of lentiviral vectors (Fig. 5a). This allows selection of the optimal promoter for a given cell system; for example, comparison of MSCV LTR and β-actin promoters driving the Arr2 miR-shRNA in RAW cells shows the former promotes more potent knockdown (Fig. 5b). Alternatively, the entry vector promoter can be removed after shRNA validation for recombination to a variety of expression platforms with existing promoters. These include 5'LTR-driven retroviral vectors or CMV/EF1-driven mammalian expression vectors which can then be used for creation of stable cell lines. These latter vectors also contain a broad selection of co-expressed fluorescent and/or drug selection markers (Additional file 1). Furthermore, the Gateway compatibility of these vectors allows the miR-shRNA cassettes to be recombined to any appropriately configured Gateway-ready expression system.

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