Described by some as
"the most important and exciting breakthrough of the last
decade" (1), RNA Inteference or RNAi is rapidly
becoming an important molecular tool for genetic research. Researchers can
phenocopy genetic loss-of-function of a specific gene by targeting its
mRNA for silencing by RNAi. RNAi experiments can provide valuable
information about gene function without waiting for the time-consuming and
laborious creation of genetic knock-outs. RNA inteference is a conserved
phenomenon across evolution–from petunias to nematodes to
human cell culture.
This article gives a
brief history of RNA Interference studies and discusses the proposed
cellular mechanism for siRNA directed interference in mammalian cells.
The first hints of RNA silencing of specific genes appeared
in 1990 when Rich Jorgensen was attempting to engineer petunias with more
intense purple color by introducing exogenous transgenes that unexpectedly
resulted in variegated pigmentation (2). The introduced DNA sequences somehow
affected the expression of the endogenous loci, and the phenomenon was given the
name "co-suppression". However, the phenomenon really sparked interest
among biologists studying the roundworm, Caenorhabdits elegans in the mid
1990s. In a 1995 paper, Guo and Kemphues describe the characterization of par-1,
a gene required for embryonic development in C. elegans (3). Loss
of par-1 function results in abnormal cleavage of the early embryos, and
embryos arrest development as a mass of cells. To phenocopy the par-1
phenotype, they injected antisense RNA into the gonad (where par-1 is
expressed) of wildtype roundworms and examined the progeny of these worms for
the par-1 phenotype. The antisense injections phenocopied par-1
loss of function, but so did the negative control, sense RNA injections!
They write in their paper: "Surprisingly, injection of in vitro synthesized sense RNA...also induced par-1
phenotypes at a high frequency among the progeny of injected worms. It is not
clear what accounts for this effect" (2).
The work of Guo and Kemphues to explain the phenomenon was
extended in the seminal RNAi paper by Fire et al., which demonstrated
that double-stranded RNA (dsRNA) was tenfold more potent at reducing gene
expression in C. elegans compared to sense or antisense RNAs alone (4).
Indeed Fire and colleagues have induced RNAi by feeding worms bacteria that were
engineered to express a specific dsRNA (5), and RNAi has been induced in worms
by soaking them in dsRNA (6). Since this work, RNAi has been reported in a
variety of organisms including zebrafish, planaria, hydra, fungi, Drosophila
and mammalian mouse embryo systems (7–13). These phenomena have been
collectively termed RNA silencing and appear to use a common set of proteins and
short RNAs. These processes are mechanistically similar, though not identical.
Mechanism
In most mammalian systems, the introduction of longer
dsRNAs (>30bp) induces a potent antiviral response that activates a dsRNA-activated
protein kinase, PKR, which phosphorylates eIF-2a, inducing a generalized
inhibition of translation (14). In addition dsRNA activates the 2´-5´
oligoadenylate polymerase/RNase L system and represses IkB, which can induce
cell death via apoptosis. Therefore, it was welcome news when Tuschl and
coworkers and Fire and colleagues showed that chemically synthesized short
interfering RNAs (siRNAs) could induce specific gene silencing in a wide range
of mammalian cell lines without causing the generalized decrease in global
protein synthesis and nonspecific mRNA degradation observed with the longer
dsRNAs (15,16).
RNAi using short dsRNAs occurs through a multistep process. The dsRNA is recognized
by an RNase III family member (e.g., Dicer in Drosophila) and is cleaved
into siRNAs of 21–23 nucleotides (17–19). In the next step, the siRNAs are
incorporated into an RNAi targeting complex known as RISC (RNA-induced silencing
complex), which targets mRNAs that are homologous to the integral siRNA of the
complex (18,19). The target mRNA is cleaved in the center of the region
complementary to the siRNA (17), with the net result of rapid degradation of the
target mRNA and a decrease in protein expression. The most potent siRNA duplexes
are 21 nucleotides long, comprising a 19bp sequence with a 2-uridine 3´
overhang at each end (17).
siLentGene™-2 U6 Cassette RNA Interference System:
For RNAi in Mammalian Systems
Since the discovery of the RNAi phenomenon, researchers have
been interested in using it to examine the function of specific genes. Several
tools exist for performing RNAi experiments including in vitro synthesized
siRNAs and vector-based systems for introducing siRNAs into cells. However,
these tools are either expensive or laborious. Only very limited guidelines exist for
selecting the precise target sequence for RNAi experiments, and there is no
guarantee that any given 21 nucleotide sequence will result in gene expression
knock out or knock down when it is introduced into a cell. Therefore,
three to five targets must be tried for each gene targeted, which can be
expensive if synthetic RNA oligos are ordered and laborious if the RNA is
generated from a vector-based system.
The siLentGene™ U6 Cassette RNA Interference System
(20) is a DNA cassette-based approach for creating siRNA expression constructs for direct delivery into cells in a rapid and cost-effective manner. The primer-dependent, PCR-based system precisely places selected mRNA sequences under the control of an
U6 promoter and terminator. The PCR products are directly
transfected into cells, eliminating the need to laboriously clone each one.
Relatively simple promoter and terminator sequences direct the production of large amounts of siRNA by the endogenous U6 polymerase of the transfected cells.
The siLentGene™ System is fast and inexpensive and provides a convenient approach for rapidly screening and determining the efficacy of different
siRNAs.
T7 RiboMAX™ Express RNAi System: For RNAi in
Non-Mammalian Systems
The T7 RiboMAX™ Express RNAi System (21)
is an in vitro transcription system designed for the production of milligram
amounts of dsRNA in a short amount of time. The dsRNA is free of protein and
other contaminants and is suitable for use in RNA interference (RNAi) in
non-mammalian cells. The system is designed for the synthesis of dsRNA molecules
of 200bp and larger and can be used with plasmid or PCR templates. Using this
system, two complementary RNA strands are synthesized by DNA templates supplied
by the user. The resulting RNA strands are annealed after the transcription
reaction to form dsRNA. Any remaining single-stranded RNA and DNA are removed
with a nuclease digestion step. The dsRNA is purified by alcohol precipitation
and can be introduced into the organism of choice for RNAi analysis.
Products may be covered by pending or issued patents or may have certain
limitations on use. Please visit
our patent and trademark web page for more information.
RiboMAX and siLentGene are trademarks of Promega Corporation.
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