Lentiviral Vector Delivery of Recombinant Small Interfering RNA Expression Cassettes

2005 ◽  
pp. 218-226 ◽  
Author(s):  
Ming-Jie Li ◽  
John J. Rossi
Keyword(s):  
Small Interfering Rna ◽  
Lentiviral Vector ◽  
Rna Expression ◽  
Interfering Rna ◽  
Vector Delivery

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 Single small-interfering RNA expression vector for silencing multiple transforming growth factor-  pathway components

2005 ◽  
Vol 33 (15) ◽  
pp. e131-e131 ◽  
Author(s):  
A. Jazag ◽  
F. Kanai ◽  
H. Ijichi ◽  
K. Tateishi ◽  
T. Ikenoue ◽  
...  
Keyword(s):  
Growth Factor ◽  
Small Interfering Rna ◽  
Expression Vector ◽  
Transforming Growth Factor ◽  
Rna Expression ◽  
Interfering Rna

scholarly journals Dynamic parent-of-origin effects on small interfering RNA expression in the developing maize endosperm

2014 ◽  
Vol 14 (1) ◽  
Author(s):  
Mingming Xin ◽  
Ruolin Yang ◽  
Yingyin Yao ◽  
Chuang Ma ◽  
Huiru Peng ◽  
...  
Keyword(s):  
Small Interfering Rna ◽  
Rna Expression ◽  
Maize Endosperm ◽  
Parent Of Origin ◽  
Origin Effects ◽  
Interfering Rna

scholarly journals Shiga Toxin Regulates Its Entry in a Syk-dependent Manner

2006 ◽  
Vol 17 (3) ◽  
pp. 1096-1109 ◽  
Author(s):  
Silje Ugland Lauvrak ◽  
Sébastien Wälchli ◽  
Tore-Geir Iversen ◽  
Hege Holte Slagsvold ◽  
Maria Lyngaas Torgersen ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Shiga Toxin ◽  
Small Interfering Rna ◽  
Dominant Negative ◽  
Target Cells ◽  
Rna Expression ◽  
Dependent Manner ◽  
Golgi Transport ◽  
Syk Inhibitor ◽  
Interfering Rna

Shiga toxin (Stx) is composed of an A-moiety that inhibits protein synthesis after translocation into the cytosol, and a B-moiety that binds to Gb3 at the cell surface and mediates endocytosis of the toxin. After endocytosis, Stx is transported retrogradely to the endoplasmic reticulum, and then the A-fragment enters the cytosol. In this study, we have investigated whether toxin-induced signaling is involved in its entry. Stx was found to activate Syk and induce rapid tyrosine phosphorylation of several proteins, one protein being clathrin heavy chain. Toxin-induced clathrin phosphorylation required Syk activity, and in cells overexpressing Syk, a complex containing clathrin and Syk could be demonstrated. Depletion of Syk by small interfering RNA, expression of a dominant negative Syk mutant (Syk KD), or treatment with the Syk inhibitor piceatannol inhibited not only Stx-induced clathrin phosphorylation but also endocytosis of the toxin. Also, Golgi transport of Stx was inhibited under all these conditions. In conclusion, our data suggest that Stx regulates its entry into target cells.

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