Effective expression of small interfering RNA in human cells
Cynthia P. Paul1, Paul D. Good1, Ira Winer2
& David R. Engelke1, 2
1
Department of Biological Chemistry, The University
of Michigan, Ann Arbor, MI
48109-0606.
2
Medical Scientist Training Program, The University
of Michigan, Ann Arbor, MI
48109-0606.
Correspondence should be addressed to David R. Engelke engelke@umich.edu
In many eukaryotes, expression of nuclear-encoded mRNA can be
strongly inhibited by the presence of a double-stranded RNA (dsRNA)
corresponding to exon sequences in the mRNA (refs 1,2). The use of this "RNA
interference" (RNAi) in mammalian studies had lagged well behind its
utility in lower animals because uninterrupted RNA duplexes longer than 30 base
pairs trigger generalized cellular responses through activation of
dsRNA-dependent protein kinases3. Recently it was
demonstrated4 that RNAi can be made to work in cultured human
cells by introducing shorter, synthetic duplex RNAs (20 base pairs)
through liposome transfection. We have explored several strategies for
expressing similar short interfering RNA (siRNA) duplexes within cells from
recombinant DNA constructs, because this might allow long-term target-gene
suppression in cells, and potentially in whole organisms. Effective suppression
of target gene product levels is achieved by using a human U6 small nuclear RNA
(snRNA) promoter to drive nuclear expression of a single RNA transcript. The
siRNA-like parts of the transcript consists of a 19−base pair siRNA stem
with the two strands joined by a tightly structured loop and a
U1−4 3' overhang at the end of the antisense strand. The
simplicity of the U6 expression cassette and its widespread transcription in
human cell types suggest that this mode of siRNA delivery could be useful for
suppressing expression of a wide range of genes.
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20 base pairs)
through liposome transfection. We have explored several strategies for
expressing similar short interfering RNA (siRNA) duplexes within cells from
recombinant DNA constructs, because this might allow long-term target-gene
suppression in cells, and potentially in whole organisms. Effective suppression
of target gene product levels is achieved by using a human U6 small nuclear RNA
(snRNA) promoter to drive nuclear expression of a single RNA transcript. The
siRNA-like parts of the transcript consists of a 19−base pair siRNA stem
with the two strands joined by a tightly structured loop and a
U1−4 3' overhang at the end of the antisense strand. The
simplicity of the U6 expression cassette and its widespread transcription in
human cell types suggest that this mode of siRNA delivery could be useful for
suppressing expression of a wide range of genes.
