Comparative analysis of DNA vectors at mediating RNAi in Anopheles mosquito cells and larvae
Department of Biological Sciences, SAF Building, Imperial College London, Imperial College Road, London SW7 2AZ, UK
* Author for correspondence (e-mail: f.catteruccia{at}ic.ac.uk)
Accepted 6 March 2003
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Summary |
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Key words: RNAi, heritable RNA interference, Anopheles, mosquito, EGFP, DNA vector, gene silencing.
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Introduction |
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To date, the only reported attempts at using RNAi to study gene function in
Anopheles mosquitoes have relied upon the direct delivery of high
concentrations of in vitro-synthesised dsRNA molecules in cell lines
and adults (Blandin et al.,
2002; Levashina et al.,
2001
). While this approach offers a rapid genetic screen of
putative target genes, interference to gene expression is transient,
non-inheritable and subject to significant variations between individuals. In
some organisms, including Caenorhabditis elegans
(Tavernarakis et al., 2000
),
trypanosomes (Bastin et al.,
2000
; Shi et al.,
2000
), Drosophila
(Fortier and Belote, 2000
;
Kennerdell and Carthew, 2000
;
Piccin et al., 2001
) and
plants (Smith et al., 2000
),
stable expression of dsRNA from integrated transgenes exhibiting dyad symmetry
has recently been achieved, thus overcoming the problems related to the
transient nature of RNAi mediated by the delivery of in
vitro-synthesised dsRNA. The stable RNAi approach is anticipated to
contribute significantly to the investigation of the function of genes
involved in mosquitoparasite interactions in Anopheles
mosquitoes.
In this study, we have assessed the molecular requirements for maximising
the silencing efficiency of dsRNA-encoding genes in Anopheles. We
have developed a series of constructs marked with the red fluorescent protein
DsRed (Matz et al., 1999), in
which sense and antisense regions of the coding sequence of the green
fluorescent protein EGFP (Heim
and Tsien, 1996
) were connected by different spacers and placed
under the control of a constitutive promoter. The silencing activity of these
constructs was assessed in A. stephensi larvae and A.
gambiae cells transiently expressing the EGFP target gene.
Furthermore, an RNAi construct was also analysed for its ability to silence an
EGFP gene stably integrated in the genome of A. stephensi
larvae.
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Materials and methods |
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Cell culture and plasmid transfections
A. gambiae Sua 4.0 cells were grown in Schneider's
Drosophila medium (GIBCO, Paisley, UK) supplemented with 10% foetal
calf serum (FCS), 100 units ml1 penicillin and 100 µg
ml1 streptomycin (Invitrogen) at 25°C. A total of
4x105 cells ml1 were plated 24 h before
co-transfection experiments, and solutions containing 4 µg each of pMinEGFP
(Fig. 1A) and either pMiRED or
a dsRNA-transcribing plasmid were co-transfected as described previously
(Catteruccia et al., 2000b).
Cells were examined two days after transfection at a wavelength of 490 nm to
detect EGFP expression and 565 nm to detect DsRed expression.
Mosquito breeding and rearing
Wild-type and transgenic Anopheles stephensi Liston adult
mosquitoes (strain sd 500) were maintained at 28°C, 70% humidity and fed
on 5% glucose. To induce egg production, female mosquitoes 35 days old
were starved for 5 h and allowed to feed on mouse blood. Two days after blood
feeding, eggs were laid and transferred into buckets with H2O
containing 5% NaCl. Larvae were grown at 25°C, 70% humidity and fed on
fish food. After 1012 days, pupae were collected and adult mosquitoes
were allowed to emerge in cages.
Embryo microinjection
A. stephensi mosquito embryos were injected essentially as
described previously (Catteruccia et al.,
2000a). For transient RNAi studies, wild-type A.
stephensi embryos were injected with a mixture of pMinEGFP (400 µg
ml1) and either pMiRED or one of the dsRNA-transcribing
plasmids (400 µg ml1). For RNAi studies against stably
expressed EGFP, A. stephensi line VB
(Catteruccia et al., 2000a
) was
injected with plasmid pIR-EGFP465Linker (400 µg
ml1). The levels of EGFP expression were assessed daily in
larvae positive for DsRed at the wavelengths described above.
Quantitative analysis of RNAi
Cells and larvae were captured on a Nikon inverted microscope with an
attached Nikon DXM1200 digital camera. Fluorescent gene expression was
quantified using the Lucia G image-processing and analysis software (Version
4.61, Nikon UK, Kingston, UK). The levels of EGFP and DsRed expression were
measured using the MeanGreen and MeanRed feature of the software, which
calculates the statistical mean of the intensity of the green or red
components of pixels, respectively. Using this software, it was possible to
calculate the mean EGFP fluorescence in only those cells in which DsRed was
co-expressed. The software was validated by measuring the intensity of EGFP
expression in homozygous and heterozygous larvae from the A.
stephensi line VB. Briefly, individuals homozygous for the
EGFP insertion consistently showed MeanGreen values that were
approximately double those from heterozygous larvae of the same age,
demonstrating a good correlation between MeanGreen values and transgene copy
number. For statistical analyses, unpaired t-tests were performed;
the null hypothesis was rejected at P0.05.
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Results |
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dsRNA-mediated gene silencing in A. gambiae Sua 4.0 cells
To assess the ability of a DNA-based system to mediate RNAi in
Anopheles cells, dsRNA-transcribing constructs, marked with the
DsRed marker gene, were co-transfected with target plasmid pMinEGFP
into the A. gambiae Sua 4.0 cell line in a series of consecutive
experiments. In this experimental system, the occurrence of RNAi against the
target gene will be indicated by a decrease in the intensity of EGFP
fluorescence in those cells that express the DsRed marker. Control experiments
were performed by co-transfecting the target construct pMinEGFP with plasmid
pMiRED, containing the DsRed marker cassette but not the EGFP-IR
(Fig. 1A). In these cells,
perfect co-localization of the green-fluorescent target and red-fluorescent
control plasmids was observed (Fig.
2A). The intensity of fluorescence of the EGFP and DsRed proteins
was not affected by the expression of the other marker (data not shown). In
all samples, quantitative analysis was performed using an image-processing
software that allowed the quantification of the mean intensity of green and
red fluorescence of each cell. In cells co-transfected with pMinEGFP and
pIR-EGFP465S, in which the A. gambiae intron was placed in
its splicing orientation, a 93.6% reduction of EGFP expression was observed as
compared with control experiments (Fig.
3A). Cells transfected with pIR-EGFP465NS, containing
the intron in its inverted, non-splicing orientation, exhibited varying
degrees of EGFP silencing in different cell subsets, ranging from
almost complete to very limited silencing and averaging 70.7% with respect to
controls (Fig. 3A). In cells
transfected with pIR-EGFP465Linker, in which the large hairpin loop
was replaced with a short 10 bp spacer region, silencing of the target gene
expression increased to 98.2% in all transfected cells (Figs
2B,
3A). High-level silencing
(96.4% inhibition) was also observed when the dsRNA corresponding to the
full-length EGFP sequence was delivered (pIR-EGFP720S;
Fig. 3A). In all samples
analysed, dsRNA transcription did not inhibit DsRed expression, and the levels
of the DsRed protein were consistently comparable to controls
(Fig. 3A).
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Transient dsRNA-mediated gene silencing in A. stephensi larvae
To examine whether the RNAi-mediated gene silencing observed in A.
gambiae cells could also be achieved in vivo, the activity of
the dsRNA constructs was analysed in mosquitoes. A. stephensi embryos
were microinjected with a mixture of the target vector pMinEGFP and one of the
dsRNA-transcribing plasmids. Larvae surviving the injection procedure were
analysed for DsRed fluorescence as an indicator of successful delivery of the
DNA species. To ascertain the perfect co-localization of target and targeting
constructs, control experiments were performed by injecting a mixture of the
pMinEGFP and pMiRED plasmids. In all control larvae analysed, the expression
of the green and red fluorescent protein largely coincided, as indicated by
their overlapping profiles (Fig.
2C). However, when pIR-EGFP465S was co-injected with
the target plasmid, a 76% decrease in the level of EGFP expression was
observed with respect to control larvae
(Fig. 3B). A similar effect was
observed after the injection of construct pIR-EGFP720S, which
caused a 79% reduction in EGFP expression
(Fig. 3B). When construct
pIR-EGFP465Linker was injected, reduction of EGFP expression was
almost complete, averaging 93% as compared with control larvae (Figs
2D,
3B). Although some EGFP
expression was observed in larvae injected with all three constructs, it
appeared to be confined to those few cells that did not exhibit expression of
the DsRed gene. In agreement with the cell data, plasmid
pIR-EGFP465NS had a less significant silencing effect, and EGFP
inhibition averaged 58% with respect to controls
(Fig. 3B).
RNAi against a stably expressed EGFP target gene
We then assessed the effects of the dsRNA-transcribing construct
pIR-EGFP465Linker on a stably expressed EGFP transgene.
This construct was chosen as it had consistently mediated the strongest
inhibition of target gene expression in both cell lines and larvae. A.
stephensi embryos from transgenic line VB, stably expressing
the EGFP gene under the control of the actin5C promoter,
were injected with pIR-EGFP465Linker, and EGFP expression was
monitored throughout larval development. As a control, homozygous embryos from
line VB were also injected with plasmid pMiRED
(Fig. 1A). Quantitative
analysis of fluorescence was performed between three days and five days
post-hatching, over which period control larvae showed a linear increase in
intensity of EGFP (Fig. 3C).
Injection of pIR-EGFP465Linker significantly decreased the overall
intensity of green fluorescent protein expression (Figs
2E,
3C), with the most noticeable
effects observed five days post-hatching when 80% silencing was achieved
(Fig. 3C). Late larval stages
showed more varied silencing effects. In some cases, EGFP expression was
slowly restored to normal levels, while in approximately 50% of larvae gene
silencing continued to be observed (data not shown). Injections were performed
into the posterior end of the embryos, thus the DsRed marker was mainly
localised in the last few segments of the larval abdomen
(Fig. 2E).
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Discussion |
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The results reported here show that inhibition of target gene expression
depended considerably upon the spacer region separating the inverted repeats,
while the length of the IR itself did not seem to significantly affect
silencing efficacy. In particular, the presence of a 67 bp-long spacer
(corresponding to the non-splicing orientation of the intron) dramatically
reduced inhibition of EGFP expression, while the same spacer in its splicing
orientation mediated high-level silencing of the target gene. While the basis
for the high efficiency of gene silencing by intron-spliced dsRNA in
vivo is not known, it is believed that the process of intron excision may
transiently increase the amount of dsRNA either by promoting its formation or
by creating a smaller, less nuclease-sensitive loop
(Smith et al., 2000).
Furthermore, the presence of an intron might increase the genomic stability of
RNAi transgenes, as genomic IR have been shown to be unstable
(Bi and Liu, 1996
;
Leach, 1994
) and increasing
the distance between the IR has been postulated to reduce their recombinogenic
potential (Lobachev et al.,
2000
). On the other hand, a long spacer may obstruct the annealing
of the IR, causing a modest number of dsRNA molecules to be formed, or make
the dsRNA molecules more nuclease sensitive. Construct
pIR-EGFP465Linker, containing the smaller spacer, consistently
mediated the highest silencing in both cells and larvae. This finding could be
explained on the basis of faster kinetics of dsRNA formation, while the
intron-containing constructs required splicing to occur before export to the
cytoplasm where they could mediate silencing. The two intron-splicing
constructs containing different IR lengths performed equally well, with no
significant differences between them. This finding seems to indicate that the
length of the dsRNA construct does not play a major role in silencing
efficiency in Anopheles.
The EGFP target gene transiently introduced into
Anopheles cells and embryos provided a simple and impartial model for
testing the gene-silencing efficacy of the targeting constructs. This
fluorescent marker allowed analysis at the protein level both visually and
quantitatively using image-processing software. RNAi was also observed when a
stably integrated EGFP gene was targeted. The analysis of RNAi
against an endogenous gene could have been compromised by the occurrence of
selection against constructs inducing a high degree of inhibition, as this
could have had a deleterious effect on fitness and viability of the organism.
The levels of EGFP inhibition depended on the amount of
pIR-EGFP465Linker delivered. In individuals injected with a 10-fold
lower concentration of pIR-EGFP465Linker, no significant silencing
of EGFP expression was observed (not shown). Plasmid DNA persisted for many
days after injection, and in most cases DsRed was still visible in fourth
instar larvae. This could represent an advantage over using in
vitro-synthesised dsRNA molecules, whose half-life has been postulated to
be too limited to study late developmental genes
(Kennerdell and Carthew, 1998;
Misquitta and Paterson, 1999
;
Montgomery and Fire, 1998
;
Wianny and Zernicka-Goetz,
2000
).
The results described here provide the first comparison of DNA constructs in mediating gene silencing in Anopheles and represent an important step towards the development of stable and heritable RNAi in these important malaria vectors.
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Acknowledgments |
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