Redesigning Animal Agriculture

(Hammond et al., 2000). Binding of the siRNA
to RISC results in the ATP-dependent activation
of this complex (Nykanen et al., 2001)
and unwinding of the siRNA. Interestingly,
a bias in the formation of active RISC with
a particular RNA strand has been observed
and is thought to reflect how the siRNAs are
processed, unwound, and loaded into RISC,
which is critically determined by the nucleotide
interactions within the siRNA (Khvorova
et al., 2003; Schwarz et al., 2003). This bias is
also evident in naturally occurring miRNAs
whose strands accumulate in RISC in unequal
proportions (Bartel, 2004). It is presumed that
this strand bias occurs by the rate-limiting
unwinding step that occurs during the transition
of the siRNA duplex to the RISC, which
allows the most weakly paired end to enter
first (reviewed in Meister and Tuschl, 2004).
Upon binding of the unwound siRNA
within RISC to the target homologous
mRNA, cleavage occurs at a single site in the
centre of the duplex region, 10 nt from the
5' end of the siRNA (Elbashir et al., 2001a).
Although this process does not require ATP
(Nykanen et al., 2001), multiple rounds of
mRNA cleavage are more efficient in the
presence of ATP (Hutvagner and Zamore,
2002). The loaded RISC complex catalyses
hydrolysis of the target RNA phosphodiester
linkage and resembles the reaction
that occurs when Dicer generates siRNA
from dsRNAs (Martinez and Tuschl, 2004;
Schwarz et al., 2004). This understanding of
the RNAi pathway has allowed researchers
to use and optimize a number of different
approaches to harness this innate pathway
for the silencing of genes of interest.
Methods used to induce RNAi
in animal cells
Long dsRNA
RNAi mediated by the introduction of
long dsRNA has been used extensively to
investigate gene function in various organisms
including planaria (Alvarado and
Newmark, 1999), hydras (Lohmann et al.,
1999), trypanosomes (Ngo et al., 1998),
Drosophila (Kennerdell and Carthew,
Transforming Livestock with Transgenics 129
1998), mosquitoes (Caplen et al., 2002), C.
elegans (Barstead, 2001) and mouse oocytes
(Svoboda et al., 2000). This technique has
been very effective, especially since the cellular
processing of long dsRNA results in the
presentation of various siRNA sequences to
the target mRNA (Fig. 8.2). In higher order
eukaryotes including mammals and birds,
instead of dsRNA, the use of synthetic
siRNAs designed to emulate natural Dicer
cleavage products has avoided induction
of dsRNA-activated immune defence pathways
and permitted researchers to exploit
the RNAi pathway for specific gene knockdown
(Elbashir et al., 2001a) (Fig. 8.2).
Synthetic siRNAs
Important developments in the design,
production and delivery of siRNAs have
seen these processes become both well
defined and highly effective, resulting in the
extremely widespread use of this technology
(Fig. 8.2). Since there is a bias in siRNA
strand selection for RISC assembly and as
siRNA duplexes are functionally asymmetric
(Schwarz et al., 2003), with only one
of the strands having the ability to trigger
RNAi, it is preferable to design an siRNA
duplex with an antisense strand that can
enter RISC. For efficient siRNA-mediated
RNAi, consideration of the siRNA sequence
is also crucial. Sequence-dependent variability
of siRNA efficacy has been shown
by siRNAs that when targeted at different
regions of the same mRNA varied markedly
in their effectiveness (Holen et al.,
2002; Miyagishi and Taira, 2002; Hemann
et al., 2003). Thus the design of the effector
molecules is of fundamental importance
to the desired outcome. Statistical analyses
of large groups of siRNA sequences have
resulted in the identification of design rules
that substantially improve the frequency of
effective sequences (Schwarz et al., 2003;
Khvorova et al., 2003; Reynolds et al., 2004;
Amarzguioui and Prydz, 2004; Ui-Tei et al.,
2004). In general, siRNAs have been 19 or
21 nt in length with two nt 3' overhangs
of uridine residues, although increased
potency has been reported using siRNAs of
up to 27 bp in length (Kim et al., 2005).