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siRNA Duplexes (sirna + duplex)

Distribution by Scientific Domains

Medical Sciences	50%

Selected Abstracts

Metabolite identification of small interfering RNA duplex by high-resolution accurate mass spectrometry

RAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 12 2008
Yan Zou
On-line liquid chromatography/electrospray ionization high-resolution mass spectrometry (LC/ESI-HRMS) using an LTQ-Orbitrap mass spectrometer was employed to investigate the metabolite profiles of a model siRNA duplex designated HBV263. The HBV263 duplex was incubated in rat and human serum and liver microsomes in vitro. The siRNA drug and its metabolites were then extracted using a liquid-liquid extraction followed by solid-phase extraction (LLE-SPE), and analyzed by LC/ESI-MS. High-resolution accurate mass data enabled differentiation between two possible metabolite sequences with a monoisotopic molecular mass difference of less than 1,Da. ProMass deconvolution software was used to provide semi-automated data processing. In vitro serum and liver microsome incubation samples afforded different metabolite patterns: the antisense strand of the duplex was degraded preferentially in rat and human serum, while the sense strand of the duplex was less stable in rat and human liver microsomes. Copyright © 2008 John Wiley & Sons, Ltd. [source]

TREM-1 promotes survival during septic shock in mice

EUROPEAN JOURNAL OF IMMUNOLOGY, Issue 2 2007
Sébastien Gibot Dr.
Abstract Triggering receptor expressed on myeloid (TREM)-1 is integral to the inflammatory response occurring during septic shock, although its precise function has yet to be determined. Here we show that in vivo silencing of TREM-1 using siRNA duplexes in a fecal peritonitis mouse model resulted in a blunted inflammatory response and increased mortality. This was associated with impaired bacterial clearance related to marked inhibition of the neutrophil oxidative burst. By contrast, TREM-1 -silenced mice were highly resistant to a lethal endotoxin challenge, while partial silencing of TREM-1 in the bacterial peritonitis model produced a significant survival benefit. These data highlight the crucial role of the TREM-1 pathway in mounting an adequate inflammatory and cytotoxic response to polymicrobial sepsis, and both the therapeutic promise and potential risks of its modulation. [source]

Sequence-specific gene silencing in murine muscle induced by electroporation-mediated transfer of short interfering RNA

THE JOURNAL OF GENE MEDICINE, Issue 1 2004
Tsunao Kishida
Abstract Background Post-genomic biomedical research requires efficient techniques for functional analyses of poorly characterized genes in living organisms. Sequence-specific gene silencing in mammalian organs may provide valuable information on the physiological and pathological roles of predicted genes in mammalian systems. Here, we attempted targeted gene knockdown in vivo in murine skeletal muscle through the electroporation-mediated transfer of short interfering RNA (siRNA). Methods siRNA duplexes corresponding to the firefly luciferase (Luc), green fluorescent protein (GFP), or glyceraldehyde-3-phosphate dehydrogenase (GAPD) genes were delivered by electroporation into the tibial muscle of normal or enhanced GFP (EGFP) transgenic mice. Plasmid vectors carrying the Luc, hRluc or ,-galactosidase (,-gal) reporter genes were also delivered. The Luc and hRluc activities in the muscle lysates were assayed. The EGFP and GAPD expression was detected by fluorescence microscopic observation and RT-PCR, respectively. Results When Luc-specific siRNA was co-delivered with the Luc expression vector into the tibial muscle, the reporter gene expression was markedly suppressed (less than 1% of the control level) for 5 days. As little as 0.05 µg of siRNA almost completely blocked the reporter gene expression from 10 µg of the plasmid. To examine whether siRNA can also suppress expression of an endogenous gene, transgenic mice carrying the EGFP gene received intramuscular transfection of a mixture of ,-gal plasmid and GFP-specific siRNA. ,-Gal-positive cells failed to express detectable levels of EGFP, while EGFP expression was not inhibited in control mice that received nonspecific siRNA. Expression of GAPD was also suppressed by the specific siRNA. Conclusions The present system may provide a useful means of phenotypic analysis of genetic information in mammalian organs for basic research as well as therapeutic molecular targeting in the post-genomic era. Copyright © 2003 John Wiley & Sons, Ltd. [source]

Role of insulin-like growth factor binding proteins in 1,,25-dihydroxyvitamin D3 -induced growth inhibition of human prostate cancer cells

THE PROSTATE, Issue 1 2005
LaMonica V. Stewart
Abstract BACKGROUND The mechanisms underlying 1,,25-dihydroxyvitamin D3 (1,25D)-induced growth inhibition of human prostate cancer cells have not been fully elucidated. To determine whether alterations in the insulin-like growth factor (IGF) signaling axis are associated with 1,25D-induced growth inhibition, we examined the ability of 1,25D to regulate expression of IGF binding proteins (IGFBPs) in human prostate cancer cell lines. METHODS Northern and Western blot analyses were used to detect 1,25D-induced alterations in IGFBP expression. Additional in vitro studies were performed to determine the role of IGFBP-3 in 1,25D-induced growth inhibition. RESULTS 1,25D decreased mRNA levels of the growth stimulatory IGFBP-2 and induced IGFBP-3 mRNA in LNCaP and C4-2 cells. 1,25D treatment also increased secreted IGFBP-3 protein levels in prostate cancer cell lines sensitive to 1,25D growth inhibition but had little effect on IGFBP-3 expression in 1,25D-resistant DU145 cells. However, recombinant IGFBP-3 had only a minor effect on LNCaP cell growth in the presence of serum. Furthermore, siRNA duplexes that reduced IGFBP-3 expression did not alter 1,25D growth inhibition in either LNCaP or PC-3 cell lines grown in serum-containing media. CONCLUSIONS Our studies indicate 1,25D-induced up-regulation of IGFBP-3 is not required for the growth inhibitory effects of 1,25D in prostate cancer cells grown in serum-containing media. © 2005 Wiley-Liss, Inc. [source]