Stable RNA interference in Spodoptera frugiperda cells by a DNA
vector-based method
Chih-Chien Lin
1
, John T.-A. Hsu
2,3
, Kai-Ling Huang
1
, Hung-Kuan Tang
2
&
Yiu-Kay Lai
1,
*
1
Department of Life Science and Institute of Biotechnology, National Tsing Hua University, Hsinchu 30013,
Taiwan, R.O.C.
2
Division of Biotechnology and Pharmaceutical Research National Health Research Institutes, Miaoli 350,
Taiwan, R.O.C.
3
Department of Chemical Engineering, National Tsing Hua University, Hsinchu 30013, Taiwan, R.O.C.
*Author for correspondence (Fax: +886-3-5715934; E-mail: yklai@life.nthu.edu.tw)
Received 21 September 2005; Revisions requested 5 October 2005; Revisions received 22 November 2005; Accepted 25 November 2005
Key words: baculovirus, double-stranded RNA, RNA interference, Spodoptera frugiperda, stable RNAi
Abstract
Double-stranded RNA (dsRNA)-mediated interference (RNAi) is a powerful tool for silencing of gene
expression in many organisms. To establish a DNA vector-based method for stable RNAi in Spodoptera
frugiperda cells (Sf9), we created a stably transfected Sf 9 cell line to express large dsRNA fragment
targeting to silence the firefly luciferase gene (luc). The luc dsRNA specifically and stably suppressed the
baculovirus-mediated luciferase expression. Thus, gene silencing in Sf 9 cells was achieved using DNA
vectors similar to the facile design described in this study.
Introduction
RNA interference (RNAi) is a powerful means
for post-transcriptional gene silencing occurring
in a wide variety of eukaryotic organisms includ-
ing Caenorhabditis elegans, Drosophila, mammals,
etc. (Chuang & Meyerowitz 2000, Zamore 2001)
and are widely utilized in functional genomic
studies. The mechanism for RNAi constitutes a
multistep process involving the generation of ac-
tive small interfering RNA (siRNA) in the cell
through the action of an RNase III endonucle-
ase, Dicer (Tomari & Zamore 2005). The result-
ing 21–23 nucleotides (nt) siRNA are then
incorporated into a silencing complex called the
RNA-induced silencing complex (RISC), which
recognizes and silences complementary homolo-
gous RNA (Tomari & Zamore 2005).
Gene silencing by RNAi can be achieved by
several different genetic techniques, such as using
chemically synthesized siRNA, in vitro tran-
scribed double-stranded RNA (dsRNA), or
DNA vector-based dsRNA (Dykxhoorn et al.
2003). Synthesized siRNA is more efficient than
dsRNA or DNA vector-based transfection and
the initiation of gene silencing by introduction of
siRNA can be immediate. However, there are
several disadvantages associated with the use of
synthesized siRNA. First, the transduction of
siRNA into cells usually leads to transient silenc-
ing effects. Second, the transfection efficiency of
siRNA would influence the silencing effects in
target cells. Furthermore, unlike plasmid DNA,
transfected siRNA is more expensive because
they need to be chemically or enzymatically syn-
thesized. To overcome these shortcomings of
transfected siRNA, the stable RNAi that trig-
gered by a DNA vector-based method has been
developed in recent years (Dykxhoorn et al.
2003).
Biotechnology Letters (2006) 28: 271–277 Ó Springer 2006
DOI 10.1007/s10529-005-5530-3