scholarly journals Comparison of different antisense strategies in mammalian cells using locked nucleic acids, 2'-O-methyl RNA, phosphorothioates and small interfering RNA

2003 ◽  
Vol 31 (12) ◽  
pp. 3185-3193 ◽  
Author(s):  
A. Grunweller
Keyword(s):  
Nucleic Acids ◽  
Mammalian Cells ◽  
Small Interfering Rna ◽  
Locked Nucleic Acids ◽  
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)

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.

scholarly journals Inducible proteolytic inactivation of OPA1 mediated by the OMA1 protease in mammalian cells

2009 ◽  
Vol 187 (7) ◽  
pp. 959-966 ◽  
Author(s):  
Brian Head ◽  
Lorena Griparic ◽  
Mandana Amiri ◽  
Shilpa Gandre-Babbe ◽  
Alexander M. van der Bliek
Keyword(s):  
Membrane Potential ◽  
Mammalian Cells ◽  
Small Interfering Rna ◽  
Mitochondrial Network ◽  
Membrane Fusion Protein ◽  
Zinc Metalloprotease ◽  
Mitochondrial Inner Membrane ◽  
Proteolytic Cascade ◽  
And Function ◽  
Interfering Rna

The mammalian mitochondrial inner membrane fusion protein OPA1 is controlled by complex patterns of alternative splicing and proteolysis. A subset of OPA1 isoforms is constitutively cleaved by YME1L. Other isoforms are not cleaved by YME1L, but they are cleaved when mitochondria lose membrane potential or adenosine triphosphate. In this study, we show that this inducible cleavage is mediated by a zinc metalloprotease called OMA1. We find that OMA1 small interfering RNA inhibits inducible cleavage, helps retain fusion competence, and slows the onset of apoptosis, showing that OMA1 controls OPA1 cleavage and function. We also find that OMA1 is normally cleaved from 60 to 40 kD by another as of yet unidentified protease. Loss of membrane potential causes 60-kD protein to accumulate, suggesting that OMA1 is attenuated by proteolytic degradation. We conclude that a proteolytic cascade controls OPA1. Inducible cleavage provides a mechanism for quality control because proteolytic inactivation of OPA1 promotes selective removal of defective mitochondrial fragments by preventing their fusion with the mitochondrial network.

scholarly journals Apoptosis and Autophagy Induction in Mammalian Cells by Small Interfering RNA Knockdown of mRNA Capping Enzymes

2008 ◽  
Vol 28 (19) ◽  
pp. 5829-5836 ◽  
Author(s):  
Chun Chu ◽  
Aaron J. Shatkin
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Mammalian Cells ◽  
Small Interfering Rna ◽  
Fluorescent Protein ◽  
Mrna Translation ◽  
Proteolytic Processing ◽  
Tunel Staining ◽  
Interfering Rna ◽  
Capping Enzymes

ABSTRACT Addition of a 5′ cap to RNA polymerase II transcripts, the first step of pre-mRNA processing in eukaryotes from yeasts to mammals, is catalyzed by the sequential action of RNA triphosphatase, guanylyltransferase, and (guanine-N-7)methyltransferase. The effects of knockdown of these capping enzymes in mammalian cells were investigated using T7 RNA polymerase-synthesized small interfering RNA and also a lentivirus-based inducible, short hairpin RNA system. Decreasing either guanylyltransferase or methyltransferase resulted in caspase-3 activation and elevated terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining characteristic of apoptosis. Induction of apoptosis was independent of p53 tumor suppressor but dependent on BAK or BAX. In addition, levels of the BH3 family member Bim increased, while Mcl-1 and Bik levels remained unchanged during apoptosis. In contrast to capping enzyme knockdown, apoptosis induced by cycloheximide inhibition of protein synthesis required BAK but not BAX. Both Bim and Mcl-1 levels decreased in cycloheximide-induced apoptosis while Bik levels were unchanged, suggesting that apoptosis in siRNA-treated cells is not a direct consequence of loss of mRNA translation. siRNA-treated BAK−/− BAX−/− double-knockout mouse embryonic fibroblasts failed to activate capase-3 or increase TUNEL staining but instead exhibited autophagy, as demonstrated by proteolytic processing of microtubule-associated protein 1 light chain 3 (LC3) and translocation of transfected green fluorescent protein-LC3 from the nucleus to punctate cytoplasmic structures.

scholarly journals Characterization of Na+/H+ exchanger NHE8 in cultured renal epithelial cells

2007 ◽  
Vol 293 (3) ◽  
pp. F761-F766 ◽  
Author(s):  
Jianning Zhang ◽  
Ion Alexandru Bobulescu ◽  
Sunita Goyal ◽  
Peter S. Aronson ◽  
Michel G. Baum ◽  
...  
Keyword(s):  
Protein Expression ◽  
Mammalian Cells ◽  
Small Interfering Rna ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Functional Characterization ◽  
Protein Levels ◽  
Primary Amino ◽  
Interfering Rna

NHE8 is expressed in the apical membrane of the proximal tubule and is predicted to be a Na+/H+ exchanger on the basis of its primary amino acid sequence. Functional characterization of native NHE8 in mammalian cells has not been possible to date. We screened a number of polarized renal cell lines for the plasma membrane Na+/H+ exchangers (NHE1, 2, 3, 4, and 8) and found only NHE1 and NHE8 transcripts in NRK cells by RT-PCR. NHE8 protein is expressed in the apical membrane of NRK cells as demonstrated by immunoblots, confocal fluorescent immunocytochemistry, and immunoelectron microscopy. NHE1, on the other hand, is expressed primarily in the basolateral membrane. Bilateral perfusion of NRK cells grown on permeable supports shows Na+/H+ exchange activity on both the apical and basolateral membranes. NHE8-specific small interfering RNA knocks down NHE8 protein expression but does not affect NHE1 protein levels. Knockdown of NHE8 protein is accompanied by a commensurate reduction in apical NHE activity, without altered basolateral NHE activity. Conversely, transfection of NHE1-specific small interfering RNA knocks down NHE1 protein expression without affecting NHE8 protein levels and reduces basolateral NHE activity without affecting apical NHE activity. NHE8 is the only apical membrane Na+/H+ exchanger in NRK cells. NHE8 activity is Na+ dependent, displaying a cooperative sigmoidal relationship, and is highly sensitive to 5-( N-ethyl- n-isopropyl)-amiloride (EIPA). NRK cells provide a useful system where NHE8 can be studied in its native environment.

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>

scholarly journals Complex Size and Surface Charge Determine Nucleic Acid Transfer by Fusogenic Liposomes

2020 ◽  
Vol 21 (6) ◽  
pp. 2244 ◽  
Author(s):  
Marco Hoffmann ◽  
Nils Hersch ◽  
Sven Gerlach ◽  
Georg Dreissen ◽  
Ronald Springer ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Surface Charge ◽  
Messenger Rna ◽  
Mammalian Cells ◽  
Nucleic Acid Delivery ◽  
Cell Cytoplasm ◽  
Delivery Methods ◽  
New Generation ◽  
Interfering Rna

Highly efficient, biocompatible, and fast nucleic acid delivery methods are essential for biomedical applications and research. At present, two main strategies are used to this end. In non-viral transfection liposome- or polymer-based formulations are used to transfer cargo into cells via endocytosis, whereas viral carriers enable direct nucleic acid delivery into the cell cytoplasm. Here, we introduce a new generation of liposomes for nucleic acid delivery, which immediately fuse with the cellular plasma membrane upon contact to transfer the functional nucleic acid directly into the cell cytoplasm. For maximum fusion efficiency combined with high cargo transfer, nucleic acids had to be complexed and partially neutralized before incorporation into fusogenic liposomes. Among the various neutralization agents tested, small, linear, and positively charged polymers yielded the best complex properties. Systematic variation of liposomal composition and nucleic acid complexation identified surface charge as well as particle size as essential parameters for cargo-liposome interaction and subsequent fusion induction. Optimized protocols were tested for the efficient transfer of different kinds of nucleic acids like plasmid DNA, messenger RNA, and short-interfering RNA into various mammalian cells in culture and into primary tissues.

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