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Interest in RNA
interference (RNAi), a phenomenon in which introduction of
dsRNA leads to gene-specific silencing, has recently exploded due
to its widespread use in gene function studies. With RNAi, researchers
can readily study gene function in a number of organisms such as Drosophila and C.
elegans by selectively suppressing the expression of a specific
gene.
Within the last year, a number of groups have reported
that transfection of 21 nt small interfering RNAs (siRNAs) can initiate
RNAi in mammalian cells (1, 2; see also "Short
Interfering RNAs (siRNAs) Can Lead to Gene-specific Silencing in
C. elegans and Mammalian Cell Lines"). The reduction in
gene expression by siRNAs, however, is transient. While this may
be suitable for many applications, stable knockdown is required for
many others.
Within several weeks at the end of April2002, seven
groups reported the development of similar plasmid vectors that can
be used to stably express functional siRNAs in mammalian cells (3-9).
Stable transfection of siRNA expression vectors led to long-term
suppression of genes to which the siRNAs were targeted. This technique
holds great promise for a number of applications, including analysis
of loss-of-function phenotypes within cell lines over long periods
of time.
The Vector for siRNA Expression
For expression of siRNAs within cells, some researchers
engineered plasmid vectors that contained either the polymerase III
H1-RNA (3), or U6 promoter (4-7), a cloning site for the stem-looped
RNA insert, and a 4-5-thymidine transcription termination signal.
The inserts were ~50 nt, with ~20 nt inverted repeats (coding for
the dsRNA stem complementary to a target gene) and ~10 nt spacers
(coding for the loop). Polymerase III promoters were chosen because
these promoters generally have well-defined initiation and stop sites
and their transcripts lack poly(A) tails. The termination signal
for these promoters is defined by 5 thymidines, and the transcript
is typically cleaved after the second uridine. Cleavage at this position
generates a 3' UU overhang in the expressed siRNA, which is similar
to the 3' overhangs of synthetic siRNAs.
In another approach, U6 promoter-driven expression
vectors were made that expressed the sense and antisense strands
of siRNAs (8-9). Upon expression, these strands presumably anneal
in vivo to produce the functional siRNAs.
Expression of siRNAs within Cells Leads to Gene-specific
Silencing
In one report (Brummelkamp, et. al), the authors
were able to specifically reduce the expression of more than 10 target
genes, including that of CDH1 and p53. In brief, vectors
containing inserts that encoded stem-looped RNAs to a specific target
gene were transiently transfected into the human breast cancer cell
line, MCF-7. After transfection, Northern and Western blots were
used to detect a reduction in expression of the endogenous gene.
When CDH1 vectors were constructed with a single mismatch to the
target, no suppression of CDH1 was observed.
Stable Expression of siRNAs Lead to Long-term Gene
Suppression
To generate stably transfected cells containing
the siRNA expression vectors, MCF-7 cells were co-transfected with
a vector containing an insert targeted to p53 and a plasmid containing
a puromycin-resistant marker (Brummelkamp, et al.). Cells selected
with puromycin were then cultured. Lowered p53 protein levels were
seen in >50% of the stably tranfected clones 2 months after transfection.
In those clones demonstrating reduced p53 protein levels after 2
months, siRNA to p53 was clearly detected by Northern analysis. Thus
silencing induced by stable transfection of the siRNA expression
constructs is long lasting.
Applications
The approach developed by these seven research
groups has enormous potential as a tool for gene function studies.
Transiently transfected and stably expressed siRNAs provide a much
quicker and simpler alternative to transgenic mice for studying loss-of-function
phenotypes in mammalian systems.
Due to the specificity of siRNA, siRNA expression
vectors also have potential applications in gene therapy. Because
siRNAs seem to be capable of distinguishing between target mRNAs
with a single base mismatch, stably expressed siRNAs may be able
to knockdown expression of mutant alleles of oncogenes or tumor
suppressor genes without affecting expression of the wild-type
allele.
References
- Elbashir, SM, Harborth, J, Lendeckel, W, Yalcin,
A, Weber, K, and Tuschl, T. (2001). Duplexes of 21-nucleotide RNAs
mediate RNA interference in cultured mammalian cells. Nature 411:494-498.
- Caplen, NJ, Parrish, S, Imani, F, Fire, A,
and Morgan, RA. (2001). Specific inhibition of gene expression
by small double-stranded RNAs in invertebrate and vertebrate systems. Proc.
Natl. Acad. Sci. USA 98(17):9742-9747.
- Brummelkamp, TR, Bernards, R, and Agami, R.
(2002). A system for stable expression of short interfering RNAs
in mammalian cells. Science 296:550-553.
- Paddison, PJ, Caudy, AA, Bernstein, E, Hannon,
GJ, and Conklin, DS. (2002). Short hairpin RNAs (shRNAs) induce
sequence-specific silencing in mammalian cells. Genes & Dev. 16:948-958.
- Paul, CP, Good, PD, Winer, I, and Engelke,
DR. (2002). Effective expression of small interfering RNA in human
cells. Nature Biotechnol. 20:505-508.
- Sui, G, Soohoo, C, Affar, E-B, Gay, F, Shi,
Y, Forrester, WC, and Shi, Y. (2002). A DNA vector-based RNAi technology
to suppress gene expression in mammalian cells. Proc. Natl.
Acad. Sci. USA 99(6):5515-5520.
- Yu, J-Y, DeRuiter, SL, and Turner, DL. (2002).
RNA interference by expression of short-interfering RNAs and hairpin
RNAs in mammalian cells. Proc. Natl. Acad. Sci. USA 99(9):6047-6052.
- Miyagishi, M, and Taira, K. (2002). U6-promoter-driven
siRNAs with four uridine 3' overhangs efficiently suppress targeted
gene expression in mammalian cells. Nature Biotechnol. 20:497-500.
- Lee, NS, Dohjima, T, Bauer, G, Li, H, Li,
M-J, Ehsani, A, Salvaterra, P, and Rossi, J. (2002). Expression
of small interfering RNAs targeted against HIV-1 rev transcripts
in human cells. Nature Biotechnol. 20:500-505.
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