Limits...
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.

Show MeSH

Related in: MedlinePlus

Stable expression of multi-miR-shRNA transcripts in a murine macrophage cell line promotes efficient knockdown of endogenous target genes. (a) Architecture of retroviral vectors (FBneo) used to stably express single or double miR-shRNAs in RAW264.7 (RAW) macrophages. (b-d) Retroviral expression of miR-shRNA can promote efficient and stable knockdown of multiple endogenous target mRNAs; Arrestin 2 and 3 (b), Grk2 and 5 (c) and Pka Cα and Cβ (d) (Data represent averages (± SEM) from at least 2 independent sample sets). (e) Western blots from Pka Cα and Cβ RAW cell lines demonstrate >95% knockdown of target proteins.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2194719&req=5

Figure 3: Stable expression of multi-miR-shRNA transcripts in a murine macrophage cell line promotes efficient knockdown of endogenous target genes. (a) Architecture of retroviral vectors (FBneo) used to stably express single or double miR-shRNAs in RAW264.7 (RAW) macrophages. (b-d) Retroviral expression of miR-shRNA can promote efficient and stable knockdown of multiple endogenous target mRNAs; Arrestin 2 and 3 (b), Grk2 and 5 (c) and Pka Cα and Cβ (d) (Data represent averages (± SEM) from at least 2 independent sample sets). (e) Western blots from Pka Cα and Cβ RAW cell lines demonstrate >95% knockdown of target proteins.

Mentions: To determine if this shRNA expression approach could be applied to less tractable cells, we created a comprehensive range of viral vectors to which miR-shRNA cassettes could be transferred by Gateway® recombination (Additional file 1). Using a retroviral vector which bicistronically expresses the miR-shRNA(s) with a neomycin resistance gene (Fig. 3a), we created stable lines of RAW264.7 murine macrophage (RAW) cells expressing single and dual miR-shRNAs against three different pairs of signaling genes; Arr2 and 3, Grk2 and 5 and the cAMP-dependent protein kinase (Pka) catalytic subunits Cα and Cβ. We observed efficient single and dual knockdown of the endogenous target genes in RAW cells using this approach (Fig. 3b–e). These data demonstrate the flexibility of the multi-miR-shRNA approach in effectively targeting three different pairs of signaling genes in a cell line which is intractable to efficient siRNA transfection. Moreover, the ability to deplete multiple genes with redundant functions is an important technical advance in the effort to dissect signaling pathways in cells such as RAW macrophages [14].


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)

Stable expression of multi-miR-shRNA transcripts in a murine macrophage cell line promotes efficient knockdown of endogenous target genes. (a) Architecture of retroviral vectors (FBneo) used to stably express single or double miR-shRNAs in RAW264.7 (RAW) macrophages. (b-d) Retroviral expression of miR-shRNA can promote efficient and stable knockdown of multiple endogenous target mRNAs; Arrestin 2 and 3 (b), Grk2 and 5 (c) and Pka Cα and Cβ (d) (Data represent averages (± SEM) from at least 2 independent sample sets). (e) Western blots from Pka Cα and Cβ RAW cell lines demonstrate >95% knockdown of target proteins.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Stable expression of multi-miR-shRNA transcripts in a murine macrophage cell line promotes efficient knockdown of endogenous target genes. (a) Architecture of retroviral vectors (FBneo) used to stably express single or double miR-shRNAs in RAW264.7 (RAW) macrophages. (b-d) Retroviral expression of miR-shRNA can promote efficient and stable knockdown of multiple endogenous target mRNAs; Arrestin 2 and 3 (b), Grk2 and 5 (c) and Pka Cα and Cβ (d) (Data represent averages (± SEM) from at least 2 independent sample sets). (e) Western blots from Pka Cα and Cβ RAW cell lines demonstrate >95% knockdown of target proteins.
Mentions: To determine if this shRNA expression approach could be applied to less tractable cells, we created a comprehensive range of viral vectors to which miR-shRNA cassettes could be transferred by Gateway® recombination (Additional file 1). Using a retroviral vector which bicistronically expresses the miR-shRNA(s) with a neomycin resistance gene (Fig. 3a), we created stable lines of RAW264.7 murine macrophage (RAW) cells expressing single and dual miR-shRNAs against three different pairs of signaling genes; Arr2 and 3, Grk2 and 5 and the cAMP-dependent protein kinase (Pka) catalytic subunits Cα and Cβ. We observed efficient single and dual knockdown of the endogenous target genes in RAW cells using this approach (Fig. 3b–e). These data demonstrate the flexibility of the multi-miR-shRNA approach in effectively targeting three different pairs of signaling genes in a cell line which is intractable to efficient siRNA transfection. Moreover, the ability to deplete multiple genes with redundant functions is an important technical advance in the effort to dissect signaling pathways in cells such as RAW macrophages [14].

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