The Design, Preparation, and Evaluation of Asymmetric Small Interfering RNA for Specific Gene Silencing in Mammalian Cells

2012 ◽  
pp. 135-152 ◽  
Author(s):  
Chanil Chang ◽  
Sun Woo Hong ◽  
Pooja Dua ◽  
Soyoun Kim ◽  
Dong-ki Lee
Keyword(s):  
Gene Silencing ◽  
Mammalian Cells ◽  
Small Interfering Rna ◽  
Specific Gene ◽  
Interfering Rna ◽  
Preparation And Evaluation

scholarly journals Small Interfering RNA–Mediated Suppression of Fas Modulate Apoptosis and Proliferation in Rat Intervertebral Disc Cells

2017 ◽  
Vol 11 (5) ◽  
pp. 686-693
Author(s):  
Jong-Beom Park ◽  
Chanjoo Park
Keyword(s):  
Gene Expression ◽  
Mammalian Cells ◽  
Small Interfering Rna ◽  
Mrna Level ◽  
Serum Deprivation ◽  
Specific Gene ◽  
Disc Cells ◽  
Fetal Bovine ◽  
Interfering Rna

<sec><title>Study Design</title><p><italic>In vitro</italic> cell culture model.</p></sec><sec><title>Purpose</title><p>To investigate the effect of small interfering RNA (siRNA) on Fas expression, apoptosis, and proliferation in serum-deprived rat disc cells.</p></sec><sec><title>Overview of Literature</title><p>Synthetic siRNA can trigger an RNA interference (RNAi) response in mammalian cells and precipitate the inhibition of specific gene expression. However, the potential utility of siRNA technology in downregulation of specific genes associated with disc cell apoptosis remains unclear.</p></sec><sec><title>Methods</title><p>Rat disc cells were isolated and cultured in the presence of either 10% fetal bovine serum (FBS) (normal control) or 0% FBS (serum deprivation to induce apoptosis) for 48 hours. Fas expression, apoptosis, and proliferation were determined. Additionally, siRNA oligonucleotides against Fas (Fas siRNA) were transfected into rat disc cells to suppress Fas expression. Changes in Fas expression were assessed by reverse transcription-polymerase chain reaction and semiquantitatively analyzed using densitometry. The effect of Fas siRNA on apoptosis and proliferation of rat disc cells were also determined. Negative siRNA and transfection agent alone (Mock) were used as controls.</p></sec><sec><title>Results</title><p>Serum deprivation increased apoptosis by 40.3% (<italic>p</italic>&lt;0.001), decreased proliferation by 45.3% (<italic>p</italic>&lt;0.001), and upregulated Fas expression. Additionally, Fas siRNA suppressed Fas expression in serum-deprived cultures, with 68.5% reduction at the mRNA level compared to the control cultures (<italic>p</italic>&lt;0.001). Finally, Fas siRNA–mediated suppression of Fas expression significantly inhibited apoptosis by 9.3% and increased proliferation by 21% in serum-deprived cultures (<italic>p</italic>&lt;0.05 for both).</p></sec><sec><title>Conclusions</title><p>The observed dual positive effect of Fas siRNA might be a powerful therapeutic approach for disc degeneration by suppression of harmful gene expression.</p></sec>

Targeted Gene Silencing by Small Interfering RNA-Based Knock-Down Technology

2004 ◽  
Vol 5 (1) ◽  
pp. 1-7 ◽  
Author(s):  
Bentham Science Publisher Zhang J. ◽  
Bentham Science Publisher Hua Z.C.
Keyword(s):  
Gene Silencing ◽  
Small Interfering Rna ◽  
Knock Down ◽  
Interfering Rna

scholarly journals Optimization Procedure for Small Interfering RNA Transfection in a 384-Well Format

2007 ◽  
Vol 12 (4) ◽  
pp. 546-559 ◽  
Author(s):  
Jason Borawski ◽  
Alicia Lindeman ◽  
Frank Buxton ◽  
Mark Labow ◽  
L. Alex Gaither
Keyword(s):  
High Throughput Screening ◽  
Mammalian Cells ◽  
Small Interfering Rna ◽  
Dna Analysis ◽  
Lentiviral Vector ◽  
Mrna Levels ◽  
Sirna Transfection ◽  
Rna Transfection ◽  
Well Assay ◽  
Interfering Rna

High-throughput screening of RNAi libraries has become an essential part of functional analysis in academic and industrial settings. The transition of a cell-based RNAi assay into a 384-well format requires several optimization steps to ensure the phenotype being screened is appropriately measured and that the signal-to-background ratio is above a certain quantifiable threshold. Methods currently used to assess small interfering RNA (siRNA) efficacy after transfection, including quantitative PCR or branch DNA analysis, face several technical limitations preventing the accurate measurement of mRNA levels in a 384-well format. To overcome these difficulties, the authors developed an approach using a viral-based transfection system that measures siRNA efficacy in a standardized 384-well assay. This method allows measurement of siRNA activity in a phenotypically neutral manner by quantifying the knockdown of an exogenous luciferase gene delivered by a lentiviral vector. In this assay, the efficacy of a luciferase siRNA is compared to a negative control siRNA across many distinct assay parameters including cell type, cell number, lipid type, lipid volume, time of the assay, and concentration of siRNA. Once the siRNA transfection is optimized as a 384-well luciferase knockdown, the biologically relevant phenotypic analysis can proceed using the best siRNA transfection conditions. This approach provides a key technology for 384-well assay development when direct measurement of mRNA knockdown is not possible. It also allows for direct comparison of siRNA activity across cell lines from almost any mammalian species. Defining optimal conditions for siRNA delivery into mammalian cells will greatly increase the speed and quality of large-scale siRNA screening campaigns. ( Journal of Biomolecular Screening 2007:546-559)

Pre-Injection of Small Interfering RNA (siRNA) Promotes c-Jun Gene Silencing and Decreases the Survival Rate of Axotomy-Injured Spinal Motoneurons in Adult Mice

2018 ◽  
Vol 65 (3) ◽  
pp. 400-410 ◽  
Author(s):  
Ying-qin Li ◽  
Fa-huan Song ◽  
Ke Zhong ◽  
Guang-yin Yu ◽  
Prince Last Mudenda Zilundu ◽  
...  
Keyword(s):  
Survival Rate ◽  
Gene Silencing ◽  
Small Interfering Rna ◽  
Spinal Motoneurons ◽  
Adult Mice ◽  
Interfering Rna

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 Antiviral Effects of Small Interfering RNA Simultaneously Inducing RNA Interference and Type 1 Interferon in Coxsackievirus Myocarditis

2012 ◽  
Vol 56 (7) ◽  
pp. 3516-3523 ◽  
Author(s):  
Jeonghyun Ahn ◽  
Ara Ko ◽  
Eun Jung Jun ◽  
Minah Won ◽  
Yoo Kyum Kim ◽  
...  
Keyword(s):  
Rna Interference ◽  
Small Interfering Rna ◽  
Antiviral Treatment ◽  
Coxsackievirus B3 ◽  
Hydroxyl Group ◽  
Specific Gene ◽  
Type I ◽  
Antiviral Effects ◽  
Direct Demonstration ◽  
Interfering Rna

ABSTRACTAntiviral therapeutics are currently unavailable for treatment of coxsackievirus B3, which can cause life-threatening myocarditis. A modified small interfering RNA (siRNA) containing 5′-triphosphate, 3p-siRNA, was shown to induce RNA interference and interferon activation. We aimed to develop a potent antiviral treatment using CVB3-specific 3p-siRNA and to understand its underlying mechanisms. Virus-specific 3p-siRNA was superior to both conventional virus-specific siRNA with an empty hydroxyl group at the 5′ end (OH-siRNA) and nonspecific 3p-siRNA in decreasing viral replication and subsequent cytotoxicity. A single administration of 3p-siRNA dramatically attenuated virus-associated pathological symptoms in mice with no signs of toxicity, and their body weights eventually reached the normal range. Myocardial inflammation and fibrosis were rare, and virus production was greatly reduced. A nonspecific 3p-siRNA showed relatively less protective effect under identical conditions, and a virus-specific OH-siRNA showed no protective effects. We confirmed that virus-specific 3p-siRNA simultaneously activated target-specific gene silencing and type I interferon signaling. We provide a clear proof of concept that coxsackievirus B3-specific 3p-siRNA has 2 distinct modes of action, which significantly enhance antiviral activities with minimal organ damage. This is the first direct demonstration of improved antiviral effects with an immunostimulatory virus-specific siRNA in coxsackievirus myocarditis, and this method could be applied to many virus-related diseases.

scholarly journals Carbon Dots for Efficient Small Interfering RNA Delivery and Gene Silencing in Plants

2020 ◽  
Vol 184 (2) ◽  
pp. 647-657 ◽  
Author(s):  
Steven H. Schwartz ◽  
Bill Hendrix ◽  
Paul Hoffer ◽  
Rick A. Sanders ◽  
Wei Zheng
Keyword(s):  
Gene Silencing ◽  
Carbon Dots ◽  
Small Interfering Rna ◽  
Interfering Rna

scholarly journals Switching the intracellular pathway and enhancing the therapeutic efficacy of small interfering RNA by auroliposome

2020 ◽  
Vol 6 (30) ◽  
pp. eaba5379 ◽  
Author(s):  
Md. Nazir Hossen ◽  
Lin Wang ◽  
Harisha R. Chinthalapally ◽  
Joe D. Robertson ◽  
Kar-Ming Fung ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Gold Nanoparticles ◽  
Gene Silencing ◽  
Small Interfering Rna ◽  
Sirna Delivery ◽  
Delivery Systems ◽  
Ovarian Cancer Cells ◽  
Interfering Rna

Gene silencing using small-interfering RNA (siRNA) is a viable therapeutic approach; however, the lack of effective delivery systems limits its clinical translation. Herein, we doped conventional siRNA-liposomal formulations with gold nanoparticles to create “auroliposomes,” which significantly enhanced gene silencing. We targeted MICU1, a novel glycolytic switch in ovarian cancer, and delivered MICU1-siRNA using three delivery systems—commercial transfection agents, conventional liposomes, and auroliposomes. Low-dose siRNA via transfection or conventional liposomes was ineffective for MICU1 silencing; however, in auroliposomes, the same dose gave >85% gene silencing. Efficacy was evident from both in vitro growth assays of ovarian cancer cells and in vivo tumor growth in human ovarian cell line—and patient-derived xenograft models. Incorporation of gold nanoparticles shifted intracellular uptake pathways such that liposomes avoided degradation within lysosomes. Auroliposomes were nontoxic to vital organs. Therefore, auroliposomes represent a novel siRNA delivery system with superior efficacy for multiple therapeutic applications.

scholarly journals A Role for Fis1 in Both Mitochondrial and Peroxisomal Fission in Mammalian Cells

2005 ◽  
Vol 16 (11) ◽  
pp. 5077-5086 ◽  
Author(s):  
Annett Koch ◽  
Yisang Yoon ◽  
Nina A. Bonekamp ◽  
Mark A. McNiven ◽  
Michael Schrader
Keyword(s):  
Mammalian Cells ◽  
Small Interfering Rna ◽  
Transmembrane Domain ◽  
Mitochondrial Fission ◽  
Ectopic Expression ◽  
Mammalian Homologue ◽  
Necessary And Sufficient ◽  
Interfering Rna ◽  
Peroxisome Division

The mammalian dynamin-like protein DLP1/Drp1 has been shown to mediate both mitochondrial and peroxisomal fission. In this study, we have examined whether hFis1, a mammalian homologue of yeast Fis1, which has been shown to participate in mitochondrial fission by an interaction with DLP1/Drp1, is also involved in peroxisomal growth and division. We show that hFis1 localizes to peroxisomes in addition to mitochondria. Through differential tagging and deletion experiments, we demonstrate that the transmembrane domain and the short C-terminal tail of hFis1 is both necessary and sufficient for its targeting to peroxisomes and mitochondria, whereas the N-terminal region is required for organelle fission. hFis1 promotes peroxisome division upon ectopic expression, whereas silencing of Fis1 by small interfering RNA inhibited fission and caused tubulation of peroxisomes. These findings provide the first evidence for a role of Fis1 in peroxisomal fission and suggest that the fission machinery of mitochondria and peroxisomes shares common components.

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