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: 2.19). 02/2007; 8(1):98. DOI: 10.1186/1471-2199-8-98
Source: PubMed


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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    • "The ability to utilize a lentivirus provides great prospects for the establishment of transgenic schistosome strains. Furthermore, the availability of the miRNA pathway transduction enables many useful features of the system to be employed, as shown in mammalian cells and animal models (Dickins et al., 2005, 2007; McBride et al., 2008; Zhu et al., 2007), to investigate schistosome biology. Together, the lentiviral delivery of miRNA expression cassettes might allow stable gene knockdown through multiple generations of S. mansoni, opening up a new research avenue to investigate hosteparasite interactions in vivo. "
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    ABSTRACT: Schistosomiasis is a prevalent, socioeconomically important disease of humans caused by parasites of the genus Schistosoma (schistosomes or blood flukes). Currently, more than 200million people worldwide are infected with schistosomes. Despite major research efforts, there is only one drug routinely used for effective treatment, and no vaccine is available to combat schistosomiasis. The purpose of the present article is to (1) provide a background on the parasites and different forms of disease; (2) describe key immunomolecular aspects of disease induced in the host; and (3) critically appraise functional genomic methods employed to explore parasite biology, parasite-host interactions and disease at the molecular level. Importantly, the article also describes the features and advantages of lentiviral delivery of artificial microRNAs to silence genes. It also discusses the first successful application of such an approach in schistosomes, in order to explore the immunobiological role of selected target proteins known to be involved in egg-induced disease. The lentiviral transduction system provides exciting prospects for future, fundamental investigations of schistosomes, and is likely to have broad applicability to other eukaryotic pathogens and infectious diseases. The ability to achieve effective and stable gene perturbation in parasites has major biotechnological implications, and might facilitate the development of radically new methods for the treatment and control of parasitic diseases. Copyright © 2015 Elsevier Ltd. All rights reserved.
    Full-text · Article · Apr 2015 · Advances in Parasitology
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    • "The resulting ''shRNAmir'' structures serve as natural substrates in miRNA biogenesis pathways and can trigger potent knockdown, as has been demonstrated for a number of miRNA backbones, including miR-30, miR-155, and miR17-92 (Chung et al., 2006; Liu et al., 2008; Zeng et al., 2002). Although studies comparing knockdown efficacies of simple stem-loop and miRNA-embedded shRNAs have reported conflicting results (Boden et al., 2004; Boudreau et al., 2008; Silva et al., 2005), shRNAmir-based systems offer several technical advantages: (1) Like endogenous miRNAs, shRNAmirs can be expressed from Pol-II promoters, which has enabled tissue-specific RNAi studies (Hinterberger et al., 2010) as well as the development of robust tetracycline (Tet)-regulated RNAi systems (Dickins et al., 2005; Stegmeier et al., 2005); (2) in Pol-II transcripts , shRNAmirs can be placed in the 3 0 UTR of a reporter to directly monitor shRNA expression (Premsrirut et al., 2011; Stegmeier et al., 2005; Zuber et al., 2011a); (3) like miRNAs, multiple shRNAmirs can be expressed as a polycistron, providing a setup for combinatorial RNAi studies (Chung et al., 2006; Zhu et al., 2007); (4) shRNAmirs are less prone to cause toxicities by interfering with endogenous miRNA pathways (Boudreau et al., 2009; Castanotto et al., 2007; McBride et al., 2008; Premsrirut et al., 2011); and (5) the natural loop configuration of shRNAmirs such as miR-30 complies with a recently discovered ''loop-counting rule'' (Gu et al., 2012) that ensures precise Dicer cleavage and reduces off-target effects. "
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    ABSTRACT: Short hairpin RNA (shRNA) technology enables stable and regulated gene repression. For establishing experimentally versatile RNAi tools and minimizing toxicities, synthetic shRNAs can be embedded into endogenous microRNA contexts. However, due to our incomplete understanding of microRNA biogenesis, such "shRNAmirs" often fail to trigger potent knockdown, especially when expressed from a single genomic copy. Following recent advances in design of synthetic shRNAmir stems, here we take a systematic approach to optimize the experimental miR-30 backbone. Among several favorable features, we identify a conserved element 3' of the basal stem as critically required for optimal shRNAmir processing and implement it in an optimized backbone termed "miR-E", which strongly increases mature shRNA levels and knockdown efficacy. Existing miR-30 reagents can be easily converted to miR-E, and its combination with up-to-date design rules establishes a validated and accessible platform for generating effective single-copy shRNA libraries that will facilitate the functional annotation of the genome.
    Full-text · Article · Dec 2013 · Cell Reports
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    • "(a) Schematic representation of the pSLIK-PTEN lentiviral vector for Tetinducible expression of PTEN. The original pSLIK vector (Shin et al., 2006; Zhu et al., 2007) was modified to confer blasticidine resistance as described in Supplementary Information. (b) Immunoblot analysis of total cell lysates from PTEN deficient GBM tumor cell cultures (T4 & T5) re-expressing PTEN probed for cleaved PARP. "
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    ABSTRACT: Glioblastoma multiforme (GBM) is an aggressive brain tumor for which there is no cure. Overexpression of wild-type epidermal growth factor receptor (EGFR) and loss of the tumor suppressor genes Ink4a/Arf and PTEN are salient features of this deadly cancer. Surprisingly, targeted inhibition of EGFR has been clinically disappointing, demonstrating an innate ability for GBM to develop resistance. Efforts at modeling GBM in mice using wild-type EGFR have proven unsuccessful to date, hampering endeavors at understanding molecular mechanisms of therapeutic resistance. Here, we describe a unique genetically engineered mouse model of EGFR-driven gliomagenesis that uses a somatic conditional overexpression and chronic activation of wild-type EGFR in cooperation with deletions in the Ink4a/Arf and PTEN genes in adult brains. Using this model, we establish that chronic activation of wild-type EGFR with a ligand is necessary for generating tumors with histopathological and molecular characteristics of GBMs. We show that these GBMs are resistant to EGFR kinase inhibition and we define this resistance molecularly. Inhibition of EGFR kinase activity using tyrosine kinase inhibitors in GBM tumor cells generates a cytostatic response characterized by a cell cycle arrest, which is accompanied by a substantial change in global gene expression levels. We demonstrate that an important component of this pattern is the transcriptional activation of the MET receptor tyrosine kinase and that pharmacological inhibition of MET overcomes the resistance to EGFR inhibition in these cells. These findings provide important new insights into mechanisms of resistance to EGFR inhibition and suggest that inhibition of multiple targets will be necessary to provide therapeutic benefit for GBM patients.
    Full-text · Article · Oct 2011 · Oncogene
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