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Current Applications in
RNAi
DNA-directed
RNAi: Enabling Multiple Applications of RNA Interference
By Chandu Ammini , David Suhy , Sara Cunningham
RNA interference (RNAi) is the latest and most
promising technology in the biotechnology tool kit for
gene silencing. Gene silencing can be induced either by
direct administration of small interfering RNA (siRNA)
or by using DNA-directed RNAi (ddRNAi) that enables the
in vivo production of siRNAs. The ddRNAi method now is a
widely used platform for targeted drug discovery,
molecular diagnostics and the creation of diverse
molecular reagents and animal disease models, and it
offers a number of advantages over siRNA. These
advantages principally include long-term expression and
the ability to multiplex expression of RNAi molecules
from a single construct to silence multiple targets, as
well as to enable novel applications using stem cells
and in the creation of transgenic mice. This review will
cover the current status of ddRNAi technology, its
various applications and major players in each
application area.
Effective
Delivery of siRNA Targeted against Human Papillomavirus
Oncogenes
By Trisha M. Wise-Draper , Susanne I. Wells
Small inhibitory RNAs (siRNAs) have proven to be
valuable tools for knocking down the expression of
specific genes. siRNAs exhibit a high degree of
specificity and have important medical implications,
such as selective oncogene repression in cancer (1). Our
research is focused on studies of human papillomaviruses
(HPVs) and their roles in cervical carcinogenesis. A
subset of the so-called high-risk HPVs is etiologically
associated with cervical cancer, with over 97% of such
cancers testing positive for high-risk HPV genomic DNA
(2). The viral oncogenes E6 and E7 are expressed in all
cells of the primary and metastatic tumor, and sustained
E6/E7 expression is required for carcinogenesis. The E6
proteins encoded by the high-risk HPVs are known to
degrade the p53 tumor suppressor, while the E7 proteins
inhibit and degrade the retinoblastoma (Rb) tumor
suppressor family proteins.
Real-time
PCR: Advancing RNA Interference and MicroRNA Studies
By Caifu Chen , Kelly McDonald , Ada H. Wong ,
Olga V. Petrauskene , Manohar R. Furtado
In recent years, researchers have made great strides
toward understanding the role of endogenous small RNAs (miRNAs)
in gene regulation and harnessing the power of exogenous
small RNAs (siRNAs and shRNAs) as laboratory tools to
manipulate messenger RNA (mRNA) levels. As the field has
evolved, methods to identify, quantify and regulate
these RNAs also have evolved. Now, a new miRNA assay
that employs stem-loop primers allows researchers to
easily and effectively assess small RNAs and greatly
enhances the power of quantitative real-time PCR for
small RNA studies.
New
Technologies to Accelerate Small-molecule Screening of
Cell Signaling Pathways
By Michael O'Grady , George Hansen , Brian Pollok
, Debasish Raha , Shelley Hough , Kristin Wiederholt ,
Michaeline Bunting , Peter Welch
We have utilized a functional cell-based reporter
system to inter-rogate cell signaling, from target
knockdown through compound screening and verification in
biochemical assays. Small-molecule protein kinase
inhibitors were analyzed for their efficacy and potency
against components of the epidermal growth factor
receptor (EGFR) signaling pathway.
Chemical
Modifications of Synthetic siRNA
By David V. Morrissey , Shawn P. Zinnen , Brent
A. Dickinson , Kristi Jensen , James A. McSwiggen ,
Chandra Vargeese , Barry Polisky
The gene silencing activity of short interfering RNA
(siRNA) offers the potential for the development of
novel therapeutics that utilize the endogenous cellular
RNAi mechanism. To be effective, exogenously delivered
siRNA therapeutics require chemical modifications to
confer resistance to nuclei present in plasma and target
tissues. We evaluated the effects of chemical
modifications on synthetic siRNA stability and function
using 2'-fluoro, 2'-OMe and 2'-deoxy sugars and terminus
capping chemistries. The silencing activity of modified
siRNAs targeted to hepatitis B virus RNA was evaluated
in a cell culture system. Resistance to nuclease
degradation was assessed in human or mouse serum and in
human or mouse liver extracts. Chemically-modified
siRNAs completely lacking 2'OH residues demonstrate
increased human serum stability (t1/2=39-408 h) and
confer potent levels of silencing activity.
Effective
Delivery of Functional siRNAs into Cells
By Dmitriy Ovcharenko , Rich Jarvis , Kevin
Kelnar , David Brown
Scientists performing RNA interference (RNAi)
experiments in mammalian systems must deliver small
interfering RNAs (siRNAs) into cells with minimal cell
mortality. Lipid-based transfection reagents that
typically are used for siRNA delivery into immortalized
cell lines tend to be inefficient for siRNA delivery
into most primary and neuronal cell types, as well as
with cells grown in suspension. This article
demonstrates the use of electroporation to successfully
deliver siRNAs into primary cells, neuronal cell types,
cells grown in suspension and other hard-to-transfect
cultures.
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