scholarly journals Transfer and Expression of Small Interfering RNAs in Mammalian Cells Using Lentiviral Vectors

Acta Naturae ◽  
2013 ◽  
Vol 5 (2) ◽  
pp. 7-18 ◽  
Author(s):  
T. D. Lebedev ◽  
P. V. Spirin ◽  
V. S. Prassolov
Keyword(s):  
Rna Interference ◽  
Mammalian Cells ◽  
Lentiviral Vectors ◽  
Cell Delivery ◽  
Small Interfering Rnas ◽  
Shrna Expression ◽  
Widespread Application ◽  
Efficient Gene ◽  
Interfering Rna

RNA interference is a convenient tool for modulating gene expression. The widespread application of RNA interference is made difficult because of the imperfections of the methods used for efficient target cell delivery of whatever genes are under study. One of the most convenient and efficient gene transfer and expression systems is based on the use of lentiviral vectors, which direct the synthesis of small hairpin RNAs (shRNAs), the precursors of siRNAs. The application of these systems enables one to achieve sustainable and long-term shRNA expression in cells. This review considers the adaptation of the processing of artificial shRNA to the mechanisms used by cellular microRNAs and simultaneous expression of several shRNAs as potential approaches for producing lentiviral vectors that direct shRNA synthesis. Approaches to using RNA interference for the treatment of cancer, as well as hereditary and viral diseases, are under active development today. The improvement made to the methods for constructing lentiviral vectors and the investigation into the mechanisms of processing of small interfering RNA allow one to now consider lentiviral vectors that direct shRNA synthesis as one of the most promising tools for delivering small interfering RNAs.

scholarly journals Optimizing RNA interference for application in mammalian cells

2004 ◽  
Vol 380 (3) ◽  
pp. 593-603 ◽  
Author(s):  
René H. MEDEMA
Keyword(s):  
Rna Interference ◽  
Gene Silencing ◽  
Mammalian Cells ◽  
Reverse Genetics ◽  
Mutational Analysis ◽  
Life Sciences ◽  
Novel Technologies ◽  
Genome Wide ◽  
Technological Developments ◽  
Interfering Rna

Over the last 2 years, the scientific community has rapidly embraced novel technologies that allow gene silencing in vertebrates. Ease of application, cost effectiveness and the possibilities for genome-wide reverse genetics have quickly turned this approach into a widely accepted, almost mandatory asset for a self-respecting laboratory in life sciences. This review discusses some of the recent technological developments that allow the application of RNAi (RNA interference) in mammalian cells. In addition, the advantages of applying RNAi to study cell cycle events and the emerging approaches to perform mutational analysis by complementation in mammalian cells are evaluated. In addition, common pitfalls and drawbacks of RNAi will be reviewed, as well as the possible ways to get around these shortcomings of gene silencing by small interfering RNA.

scholarly journals Conditional Suppression of Cellular Genes: Lentivirus Vector-Mediated Drug-Inducible RNA Interference

2003 ◽  
Vol 77 (16) ◽  
pp. 8957-8951 ◽  
Author(s):  
Maciej Wiznerowicz ◽  
Didier Trono
Keyword(s):  
Rna Interference ◽  
Mammalian Cells ◽  
Developmental Genetics ◽  
Present System ◽  
Small Interfering Rnas ◽  
Lentivirus Vector ◽  
Molecular Therapeutics ◽  
Cellular Genes ◽  
Conditional Suppression

ABSTRACT RNA interference has emerged as a powerful technique to downregulate the expression of specific genes in cells and in animals, thus opening new perspectives in fields ranging from developmental genetics to molecular therapeutics. Here, we describe a method that significantly expands the potential of RNA interference by permitting the conditional suppression of genes in mammalian cells. Within a lentivirus vector background, we subjected the polymerase III promoter-dependent production of small interfering RNAs to doxycycline-controllable transcriptional repression. The resulting system can achieve the highly efficient and completely drug-inducible knockdown of cellular genes. As lentivirus vectors can stably transduce a wide variety of targets both in vitro and in vivo and can be used to generate transgenic animals, the present system should have broad applications.

Differential Sensitivity of Normal and CML-Derived CD34+ Cells to Inhibition of SHP-2 Gene Expression by RNA Interference (RNAi).

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2778-2778
Author(s):  
Michaela Scherr ◽  
Karin Battmer ◽  
Anuhar Chaturvedi ◽  
Beate Schultheis ◽  
Arnold Ganser ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Interference ◽  
Functional Genomics ◽  
Cell Lines ◽  
Mammalian Cells ◽  
Chronic Phase ◽  
Differential Sensitivity ◽  
Shrna Expression ◽  
Gm Csf ◽  
Cd34 Cells

Abstract RNA interference (RNAi) has rapidly evolved into an efficient tool for functional genomics in a variety of organisms. Stable expression of shRNA (short hairpin RNA) driven by pol III promoters upon retro- or lentiviral gene transfer can induce long-term gene silencing in mammalian cells, including human hematopoietic cells. We recently demonstrated that lentivirus mediated anti bcr-abl RNAi can specifically silence bcr-abl gene expression, inhibit oncogene driven cell proliferation, and eradicate leukemic cells depending on the dose of lentivirus-mediated shRNA expression (Scherr et al. Gene Therapy 2004). Since effective depletion requires a threshold of lentiviral integrations into target cell genomes, the risk of insertional mutagenesis may limit the therapeutic value of this approach. We therefore applied lentivirus-mediated RNAi for functional genomics in purified primary normal and CD34+ cells from chronic phase CML patients harvested at initial diagnosis. Several SHP-2 shRNAs were generated according to established rules and were functionally evaluated using a bicistronic reporter system as described earlier. Effective shRNA expression cassettes were subsequently cloned into lentiviral plasmids encoding RFP to track lentiviral transduction. Transduction of K562, U937, NB-4 and TF-1 cells with lentiviral supernatants results in a reduction of SHP-2 mRNA and protein by more than 90 %. Interestingly, anti-SHP-2 shRNA induced almost complete depletion of RFP+ cells in all four cell lines, demonstrating that SHP-2 expression is essential for proliferation and survival in these cells. We next transduced normal and CML-derived CD34+ cells with a puritiy of > 95% with control and anti-SHP-2 lentiviruses, and stimulated methylcellulose cultures of the cells with high (GM-CSF: 20 ng/ml; IL-3: 10 ng/ml) or low (GM-CSF: 0.2 ng/ml; IL-3: 0.1 ng/ml) cytokine concentrations. This assay relies on the fact that colony formation of CML-CFU is mediated by both cytokine receptor and bcr-abl signaling. Therefore differential numbers of transduced, i.e. RFP+ colonies under different cytokine stimulations reflect the role of the RNAi-target in normal or malignant CFU. Whereas anti-SHP-2 RNAi did not reduce the proliferation of normal transduced CFU (n=5), proliferation of CFU from CML patients was specifically reduced between 50 to 85 % under low cytokine concentration (n= 9). These data suggest that primary normal cells are more resistant to inhibition of SHP-2 gene expression than leukemic cell lines and CD34+ cells from CML patients and identify SHP-2 as a potential target for anti bcr-abl therapy.

scholarly journals Sustained long-term RNA interference in nucleus pulposus cells in vivo mediated by unmodified small interfering RNA

2009 ◽  
Vol 18 (2) ◽  
pp. 263-270 ◽  
Author(s):  
Teppei Suzuki ◽  
Kotaro Nishida ◽  
Kenichiro Kakutani ◽  
Koichiro Maeno ◽  
Takashi Yurube ◽  
...  
Keyword(s):  
Rna Interference ◽  
Nucleus Pulposus ◽  
Small Interfering Rna ◽  
Nucleus Pulposus Cells ◽  
Interfering Rna

scholarly journals Inhibition of Virus Production in JC Virus-Infected Cells by Postinfection RNA Interference

2004 ◽  
Vol 78 (13) ◽  
pp. 7270-7273 ◽  
Author(s):  
Yasuko Orba ◽  
Hirofumi Sawa ◽  
Hiroshi Iwata ◽  
Shinya Tanaka ◽  
Kazuo Nagashima
Keyword(s):  
Rna Interference ◽  
Mammalian Cells ◽  
Jc Virus ◽  
Virus Production ◽  
Viral Proteins ◽  
Small Interfering Rnas ◽  
Virus Infections ◽  
Marked Inhibition ◽  
Infected Cells ◽  
New Strategies

ABSTRACT RNA interference has been applied for the prevention of virus infections in mammalian cells but has not succeeded in eliminating infections from already infected cells. We now show that the transfection of JC virus-infected SVG-A human glial cells with small interfering RNAs that target late viral proteins, including agnoprotein and VP1, results in a marked inhibition both of viral protein expression and of virus production. RNA interference directed against JC virus genes may thus provide a basis for the development of new strategies to control infections with this polyomavirus.

scholarly journals The 3A protein of coxsackievirus B3 acts as a viral suppressor of RNA interference

2020 ◽  
Vol 101 (10) ◽  
pp. 1069-1078
Author(s):  
Jingfang Mu ◽  
Haobo Zhang ◽  
Tao Li ◽  
Ting Shu ◽  
Yang Qiu ◽  
...  
Keyword(s):  
Rna Interference ◽  
Mammalian Cells ◽  
Mutational Analysis ◽  
Nonstructural Protein ◽  
Viral Myocarditis ◽  
Coxsackievirus B3 ◽  
Small Interfering Rnas ◽  
The Family ◽  
Viral Suppressors

RNA interference (RNAi) is a potent antiviral defence mechanism in eukaryotes, and numerous viruses have been found to encode viral suppressors of RNAi (VSRs). Coxsackievirus B3 (CVB3) belongs to the genus Enterovirus in the family Picornaviridae, and has been reported to be a major causative pathogen for viral myocarditis. Despite the importance of CVB3, it is unclear whether CVB3 can also encode proteins that suppress RNAi. Here, we showed that the CVB3 nonstructural protein 3A suppressed RNAi triggered by either small hairpin RNAs (shRNAs) or small interfering RNAs (siRNAs) in mammalian cells. We further uncovered that CVB3 3A interacted directly with double-stranded RNAs (dsRNAs) and siRNAs in vitro. Through mutational analysis, we found that the VSR activity of CVB3 3A was significantly reduced by mutations of D24A/L25A/L26A, Y37A/C38A and R60A in conserved residues. In addition, the 3A protein encoded by coxsackievirus B5 (CVB5), another member of Enterovirus, also showed VSR activity. Taken together, our findings showed that CVB3 3A has in vitro VSR activity, thereby providing insights into the pathogenesis of CVB3 and other enteroviruses.

scholarly journals Elongation of Peroxisomes as an Indicator for Efficient Dynamin-like Protein 1 Knock Down in Mammalian Cells

2005 ◽  
Vol 53 (8) ◽  
pp. 1037-1040 ◽  
Author(s):  
Antje Boll ◽  
Michael Schrader
Keyword(s):  
Rna Interference ◽  
Gene Silencing ◽  
Mammalian Cells ◽  
Morphological Changes ◽  
Knock Down ◽  
Efficient Gene ◽  
Biochemical Studies ◽  
Number Of Cells

RNA interference has become a valuable tool to identify and investigate proteins involved in the formation of peroxisomes. We demonstrate that the elongation of peroxisomes serves as an excellent indicator for efficient knock down of dynamin-like protein 1 (DLP1) in mammalian cells. We took advantage of the silencing-dependent morphological changes of peroxisomes to compare different transfection methods and show that a single transfection of DLP1 siRNA by electroporation is sufficient to effectively silence DLP1. We present a fast, easy, and convenient protocol for efficient gene silencing in a large number of cells, which can be used for quantitative and biochemical studies.

Small interfering RNA treatment can inhibit Cyprinid herpesvirus 3 associated cell death in vitro

2014 ◽  
Vol 17 (4) ◽  
pp. 733-735 ◽  
Author(s):  
M. Adamek ◽  
G. Rauch ◽  
G. Brogden ◽  
D. Steinhagen
Keyword(s):  
Cell Death ◽  
Rna Interference ◽  
Small Interfering Rna ◽  
Enzyme Synthesis ◽  
Small Interfering Rnas ◽  
Viral Dna ◽  
Cyprinid Herpesvirus 3 ◽  
Interfering Rna ◽  
Fibroblastic Cells

Abstract A Cyprinid herpesvirus 3 infection of carp induces a disease which causes substantial losses in carp culture. Here we present the use of a possible strategy for the management of the virus infection RNA interference based on small interfering RNAs. As a result of in vitro studies, we found that a mixture of short interfering RNAs specific for viral DNA enzyme synthesis and capsid proteins of the CyHV-3 can be a potential inhibitor of virus replication in fibroblastic cells. This gives the basis for the development of a combinatorial RNA interference strategy to treat CyHV-3 infections.

scholarly journals A Virus-Encoded Inhibitor That Blocks RNA Interference in Mammalian Cells

2005 ◽  
Vol 79 (12) ◽  
pp. 7371-7379 ◽  
Author(s):  
Christopher S. Sullivan ◽  
Don Ganem
Keyword(s):  
Rna Interference ◽  
Mammalian Cells ◽  
Rna Virus ◽  
Small Interfering Rnas ◽  
Mechanism Of Inhibition ◽  
Highly Sensitive ◽  
Protein Functions ◽  
Dicer Cleavage ◽  
Short Hairpin Rnas

ABSTRACT Nodamura virus (NoV) is a small RNA virus that is infectious for insect and mammalian hosts. We have developed a highly sensitive assay of RNA interference (RNAi) in mammalian cells that shows that the NoV B2 protein functions as an inhibitor of RNAi triggered by either short hairpin RNAs or small interfering RNAs. In the cell, NoV B2 binds to pre-Dicer substrate RNA and RNA-induced silencing complex (RISC)-processed RNAs and inhibits the Dicer cleavage reaction and, potentially, one or more post-Dicer activities. In vitro, NoV B2 inhibits Dicer-mediated RNA cleavage in the absence of any other host factors and specifically binds double-stranded RNAs corresponding in structure to Dicer substrates and products. Its abilities to bind to Dicer precursor and post-Dicer RISC-processed RNAs suggest a mechanism of inhibition that is unique among known viral inhibitors of RNAi.

Novel lentiviral vectors displaying “early-acting cytokines” selectively promote survival and transduction of NOD/SCID repopulating human hematopoietic stem cells

Blood ◽  
2005 ◽  
Vol 106 (10) ◽  
pp. 3386-3395 ◽  
Author(s):  
Els Verhoeyen ◽  
Maciej Wiznerowicz ◽  
Delphine Olivier ◽  
Brigitte Izac ◽  
Didier Trono ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Transfer ◽  
Hematopoietic Stem Cells ◽  
Surface Display ◽  
Lentiviral Vectors ◽  
Hematopoietic Stem ◽  
Efficient Gene ◽  
Cd34 Cells

AbstractA major limitation of current lentiviral vectors (LVs) is their inability to govern efficient gene transfer into quiescent cells, such as human CD34+ cells, that reside in the G0 phase of the cell cycle and that are highly enriched in hematopoietic stem cells. This hampers their application for gene therapy of hematopoietic cells. Here, we designed novel LVs that overcome this restriction by displaying “early-acting cytokines” on their surface. Display of thrombopoietin, stem cell factor, or both cytokines on the LV surface allowed efficient gene delivery into quiescent cord blood CD34+ cells. Moreover, these surface-engineered LVs preferentially transduced and promoted survival of resting CD34+ cells rather than cycling cells. Finally, and most importantly, these novel LVs allowed superior gene transfer in the most immature CD34+ cells as compared to conventional LVs, even when the latter vectors were used to transduce cells in the presence of recombinant cytokines. This was demonstrated by their capacity to promote selective transduction of CD34+ cell in in vitro derived long-term culture-initiating cell (LTC-IC) colonies and of long-term NOD/SCID repopulating cells (SRCs) in vivo.

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