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J. Biol. Chem., Vol. 277, Issue 43, 40839-40843, October 25, 2002
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,,,,,
From the Case Western Reserve University,
School of Medicine, Department of Genetics, Cleveland, Ohio
44106-4955, § Chromatin Inc., Chicago, Illinois, 60612, and
¶ Department of Biological Sciences, SAF Building, Imperial
College of Science, Technology and Medicine, Imperial College Road,
London SW7 2AZ, United Kingdom
Received for publication, July 3, 2002, and in revised form, August 7, 2002
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Malaria kills millions of people every year, and
new control measures are urgently needed. The recent demonstration that
(effector) genes can be introduced into the mosquito germ line to
diminish their ability to transmit the malaria parasite offers new hope toward the fight of the disease (Ito, J., Ghosh, A., Moreira, L. A., Wimmer, E. A. & Jacobs-Lorena, M. (2002)
Nature, 417, 452-455). Because of the high selection
pressure that an effector gene imposes on the parasite population,
development of resistant strains is likely to occur. In search of
additional antiparasitic effector genes, we have generated transgenic
Anopheles stephensi mosquitoes that express the bee venom
phospholipase A2 (PLA2) gene from the gut-specific and
blood-inducible Anopheles gambiae
carboxypeptidase (AgCP) promoter. Northern blot
analysis indicated that the PLA2 mRNA is specifically expressed in
the guts of transgenic mosquitoes with peak expression at ~4 h after
blood ingestion. Western blot and immunofluorescence analyses detected
PLA2 protein in the midgut epithelia of transgenic mosquitoes from 8 to
24 h after a blood meal. Importantly, transgene expression reduced
Plasmodium berghei oocyst formation by 87% on average and
greatly impaired transmission of the parasite to naive mice. The
results indicate that PLA2 may be used as an additional effector gene
to block the development of the malaria parasite in mosquitoes.
Worldwide mortality due to malaria has increased in the past
decade mainly because of parasite and mosquito resistance to drugs and
insecticides, respectively, and the lack of effective vaccines (1).
Genetically engineering mosquito vectors for refractoriness to malaria
parasites is a strategy for reducing disease transmission that should
be explored. Plasmodium, the causative agent of malaria, has
to complete a complex developmental program in the mosquito for
transmission to occur. The first interactions between the parasite and
the mosquito occur in the midgut lumen, where the parasite has to
traverse two barriers, the peritrophic matrix, and the midgut
epithelium (2, 3). Because the gut is a closed compartment that limits
diffusion, antimalarial compounds secreted into the midgut lumen are
expected to efficiently target the initial stages of parasite development.
Previous studies have demonstrated that venom phospholipases A2
(PLA2s)1 strongly inhibit
oocyst formation when administered to mosquitoes with an infectious
blood meal (4). Although the mechanism of inhibition has not been
established, it is known that PLA2 does not kill ookinetes and does not
interfere with their development in vitro. Furthermore,
inhibition of oocyst formation did not depend on PLA2 enzymatic
activity. It is possible that PLA2 inhibits ookinete invasion by
modifying the properties of the midgut epithelial membranes that are
invaded by the parasite.
The best candidates for driving the expression of foreign gene products
to be secreted into the mosquito midgut are the promoters of
bloodmeal-inducible midgut genes because of their strength, tissue
specificity, and synchrony of expression with parasite ingestion by the
mosquito. In this context, we have shown that the Anopheles
gambiae and Aedes aegypti carboxypeptidase promoters can be used to drive strong expression of recombinant protein in the
midgut of transgenic mosquitoes (5) and also that they can drive the
expression of a parasite blocking peptide (6) in transgenic mosquitoes
(7). Although the latter results indicate that genetic manipulation of
mosquito vectors is a promising strategy for reducing malaria
transmission, it is also important to consider that the use of a single
effector gene is likely to lead to rapid selection of resistant
parasites. Thus, we have searched for additional effector genes whose
products interfere with parasite development by mechanisms different
from the previously developed ones (6, 7). The results reported in this
article suggest that bee venom PLA2 may be used as an
alternate effector gene.
Cloning of a Full-length Bee Venom Phospholipase A2
cDNA--
The full-length cDNA of the honeybee phospholipase
A2 gene was cloned for the first time by relying on the previously
published partial sequence (GenBankTM accession number
X16709). Messenger RNA was isolated from 105 venom glands dissected out
of newly emerged honeybees using the Micro Fast-Track mRNA
isolation kit from Invitrogen. The mRNA was reverse-transcribed
with Superscript II reverse transcriptase (Invitrogen) using the
SMART cDNA synthesis kit (Clontech) in the
presence of a primer specific to the previously published 3' end of the
honeybee PLA2 cDNA (5'-ccgccgtctagataaataaacgatctcgaagtggtactc-3'), as well as a custom-synthesized SMART primer
(5'-agcagtggtttaaacgcagagtggccattatggccggg-3'). The first strand
cDNA was PCR-amplified using the same two primers and
Platinum-Taq high fidelity polymerase (Invitrogen), and the PCR product was cloned with the TOPO-TA cloning kit (Invitrogen). The resulting construct BV PLA2 5-7 consisted of the
full-length bee venom PLA2 open reading frame flanked by 5'-
and 3'-untranslated sequences inserted into the pCR-TOPO vector
(Invitrogen). The PLA2 sequence thus obtained included the
entire signal peptide, which was only partially present in previously
published sequences, as well as a portion of the 5'-untranslated region
(UTR). Inserts from several identical clones were sequenced, and the
sequence was used to prepare PCR primers for the construction of the
mosquito expression constructs.
Carboxypeptidase-PLA2 Construct--
A 3.9-kb fragment from the
A. gambiae carboxypeptidase gene (8)
(AgCP5'; containing the promoter, 5'-UTR, and signal
peptide) was amplified by PCR from a pBluescript AgCP
genomic subclone using AgCPKpn
(5'-GGTACCCTCGGCCGCTTCGACACT-3') and T7
(5'-GTAATACGACTCACTATAGGGC-3') primers and cloned into pGemT-easy.
Bee venom PLA2 coding region (450 bp) was cloned into
pGemT-easy (Promega) from a cDNA clone using primers
PLA2K (5'-GGTACCTGGCAAATCAGGGAT-3') and PLA2B
(5'-GGATCCTTATCAATACTTGCGAAGATC-3'). The AgCP5' in
pGemT-easy was digested with KpnI, and the resulting 1.8-kb
fragment was ligated into the PLA2 plasmid
(KpnI-digested). AgCP3' (555 bp) (untranslated 3'
region) was obtained by PCR amplification with primers
AgCP3BH (5'-GGATCCTGAAGTCTCTCCTACCGG-3') or AgCP3SC (5'-CCGCGGTAAGGCTAGCATTGCCA-3') on an AgCP pBluescript
genomic subclone and cloned into pGemT-easy. Both the
AgCP/PLA2 and AgCP in pGemT were
digested with BamHI and SacII and the AgCP
3'-UTR fragment ligated to AgCP5/PLA2
(pGemT-easy). A NotI fragment containing AgCP/PLA2/AgCP3 was cloned into
pSLfa1180fa plasmid (9) that contains unique FseI
and AscI sites. The plasmid was then cut with these enzymes
and cloned into piggyBac[3xP3-EGFPafm] plasmid. The
final transposon plasmid pBac[3xP3-EGFP(AgCPPLA2)]
contains 1.7 kb of AgCP promoter, around 100 bp of
AgCP coding region to provide the signal sequence, 0.45 kb
of PLA2 coding region, and 0.58 kb of AgCP-3'UTR
apart from the EGFP sequence (see Fig. 1).
Microinjection and Mosquito Rearing--
Anopheles
stephensi embryos were injected as described (7, 10). Embryos were
injected with a mixture of the transposon construct (0.5 mg/ml) and of
the piggyBac helper plasmid (11) (0.3 mg/ml), both purified
on Qiagen midi-prep columns. No heat shock was performed. The surviving
adults were pooled into families as follows. Adult injected males
(three to five G0 mosquitoes) were crossed with 5-10
virgin non-injected females. Between three and six G1
virgin injected females were pooled and mated with three to five
non-injected males. Females were allowed to feed on blood, and
G1 embryos were collected. Transformants were selected by
screening cold-immobilized larvae for EGFP expression using a
dissecting fluorescence microscope at a wavelength of 490 nm.
Southern Blot Analysis--
Individual G1 transgenic
mosquitoes (males or females) from positive families were mated with
non-transgenic mosquitoes. Progenies of these crosses were checked for
integration of the transgene as follows. Total genomic DNA was isolated
from transgenic and wild type mosquitoes as described (12) but with 100 µg/ml proteinase K treatment at 52 °C for 3 h and followed by
phenol:chloroform extractions and precipitation. DNA digested with
BglII was separated on a 0.8% agarose gel, blotted, and
hybridized with [ Northern Blot Analysis--
Female mosquito guts and carcasses
(whole body minus gut) and male guts were dissected from 4-5-day old
adults. All tissues were immediately frozen in an ethanol/dry ice bath
and stored at Immunoblot Analysis--
Guts were dissected in
phosphate-buffered saline (PBS) at different times after a blood meal.
The equivalent of 0.4 guts/lane was analyzed by electrophoresis on a
15% polyacrylamide/SDS gel followed by electrotransfer to a
polyvinylidene fluoride membrane (Millipore). A prestained protein
ladder (Benchmark, Invitrogen) was loaded on the same gel. The membrane
was incubated with an anti-rabbit bee venom phospholipase A2 polyclonal
antibody (Accurate; 1:2,000 dilution), and the bound antibody was
detected with an anti-rabbit immunoglobulin, horseradish
peroxidase-linked (New England Biolabs, 1:3,000 dilution) by exposing
the blots to x-ray films.
Oocyst Formation Assays--
About 2 × 107
Plasmodium berghei parasites (ANKA 2.34) were inoculated per
naive mouse, and gametocyte-positive mice were used to feed a mixed
population of non-transgenic and transgenic mosquitoes. Mosquitoes that
blood-fed were separated after 24 h and kept at 21 °C with 10%
sugar solution. On day 15, midguts were dissected, and the number of
oocysts was determined.
Transmission Blocking Assays--
Transgenic and sibling
non-transgenic mosquitoes were mixed in the same container and allowed
to feed on a single infected mouse. Mosquitoes that blood-fed were
separated after 24 h and kept at 21 °C with 10% sugar
solution. On day 25, individual female mosquitoes were separated into
small feeding cups, and each mosquito was allowed to feed on a single
naive mouse (Swiss Webster (CFW), Charles River Laboratory).
After feeding, mosquitoes were cold-immobilized, salivary glands were
dissected and homogenized in a small volume of PBS, and sporozoites
were counted with a hemacytometer. The infection status of each mouse
was followed by observing tail vein blood smears on days 5, 7, 9, 13, 17, 22, and 25.
Immunofluorescence Assays of Midgut Sheets--
Female mosquito
guts were dissected at different times after a blood meal in 50%
ethanol and opened longitudinally to make a sheet. The gut sheets were
treated with a methanol series to remove the autofluorescence as
described (6), fixed overnight in 4% paraformaldehyde at 4 °C,
washed six to seven times with PBS, and blocked for 6 h with
PBS/4% bovine serum albumin/4% fetal calf serum at 4 °C. The guts
were then incubated overnight at 4 °C with an anti-rabbit bee venom
phospholipase A2 antibody (Accurate; 1:2,000 dilution). The sheets were
washed seven times and incubated in the dark for 2 h with a
fluorescein isothiocyanate-conjugated anti-rabbit IgG (Sigma, 1:600
dilution). The gut sheets were mounted onto glass slides, covered with
a drop Slowfade Antifade (Molecular Probes), and examined by
fluorescence microscopy.
Immunofluorescence Assays of Midgut Sections--
Guts from
blood-fed mosquitoes were dissected in 4% paraformaldehyde in PBS,
fixed for 2 h at room temperature, and washed three times in PBS.
After dehydration in a graded ethanol/PBS series, the guts were treated
with xylenes and embedded in Paraplast (Oxford Labware), and 7-14-µm
sections were mounted onto glass slides. The slides were washed twice
in xylenes at room temperature followed by rehydration in a graded
ethanol/PBS series. To reduce autofluorescence, slides were dehydrated
and rehydrated in a graded methanol/PBS series. The slides were blocked
in 10% nonfat milk and 0.1% Triton X-100 in PBS for 2 h at room
temperature and incubated overnight at room temperature with an
anti-bee venom phospholipase A2 antibody (Accurate; 1:300 dilution in
blocking solution). Slides were washed three times for 30 min with
blocking solution and incubated for 2 h at room temperature with
FITC-conjugated anti-rabbit antibodies (Sigma; 1:600 dilution). Nuclei
were visualized by staining with 4',6-diamidino-2-phenylindole (DAPI;
Molecular Probes) diluted with Slowfade Antifade solution.
Immunostaining was observed by fluorescence microscopy at ×400
magnification. DAPI staining was observed on the same sections by UV
illumination and merged with the FITC-fluorescence images using Adobe
PhotoShop software (Adobe Systems Inc.)
A. stephensi Transformation--
For the expression of PLA2
in the A. stephensi midgut, we
constructed the AgCP-PLA2 gene that consists of the
promoter, the 5'-UTR, and the signal peptide from the AgCP
gene (8) linked to the coding sequence of the bee venom PLA2
gene and the AgCP 3'-UTR (Fig.
1). This gene was inserted into the
pSL1180fa shuttle plasmid, which has unique restriction
sites for cloning into the 3xP3-EGFP piggyBac transformation
vector (9). This vector allows convenient screening of transformed
mosquitoes since fluorescence can easily be detected in the eyes of
both larvae and adult mosquitoes. Importantly, the dark pigments of the
adult eye do not interfere with detection of the GFP marker protein
(7). Moreover, confined GFP expression in only a few tissues may be
beneficial for mosquito fitness. piggyBac proved to be an
efficient vector for A. stephensi transformation.
Of 399 embryos injected, 58 adults were obtained and pooled into 18 families. Four different transgenic mosquito lines (AM3, AF1, BF4, and
BM1) were established from two GFP-positive families (details in the
following text). The minimum calculated transformation rate among the
surviving adults was 6.9%. By comparison, transformation rates were
7% for A. stephensi with Minos (10) and 4% for the following combinations: A. stephensi with piggyBac (14), A. aegypti with Hermes (5, 15, 16), and
A. aegypti with mariner (5, 17).
Characterization of the Transgenic Lines--
To confirm that the
transgene was stably integrated and to determine the number of
integrated transposons per genome, DNA from each mosquito line was
analyzed by Southern blot hybridization. Mosquito genomic DNA was
digested with BglII, fractionated, blotted, and hybridized
with a probe from the piggyBac left arm (Fig. 1, probe
a). As shown in Fig. 2, each
mosquito line carried a single transgene integrated at a different
position, indicating that independent integration events had occurred
in each of the four families (note that AM3 and BM1 originated from
different families). No signal was detected with DNA from
non-transformed mosquitoes (data not shown).
Expression of the AgCP-PLA2 mRNA was investigated by Northern
analysis (Fig. 3). A single band of the
expected size (~800 bases) was detected (18). The AgCP-PLA2 mRNA
was present in the midgut of sugar-fed mosquitoes and was strongly
induced by blood ingestion (peak at ~4 h), consistent with the
pattern of expression of the A. gambiae
carboxypeptidase gene (8). Enhanced expression at 48 h was also
observed, but the reasons for this are not clear. Robust mRNA
expression as well as tissue and sex specificity together indicate that
the 1.7-kb AgCP upstream sequence contains the necessary
regulatory elements.
Immunoblot analysis showed that the anti-PLA2 antibody detected a
protein that was produced in a blood-inducible manner in the midguts of
transgenic mosquitoes (Fig. 4). The
electrophoretic mobility of the protein (17 kDa) on acrylamide gels
was identical to that of an high pressure liquid
chromatography-purified bee venom PLA2 protein (data not shown). Less
than 0.5 gut equivalent of protein per lane was sufficient for PLA2
detection. Importantly, the recombinant protein showed a peak of
expression between 8 and 24 h after a blood meal, coinciding with
the time of ookinete invasion of the midgut (3). By 48 h, the
protein was not detectable anymore, which contrasts with the Northern
data.
Immunofluorescence assays detected the protein in whole-mount gut
sheets (Fig. 5) and in gut sections (Fig.
6). In sheets, the strongest signal
appeared in epithelia prepared from guts 6 h after a blood meal,
whereas in sections, a much stronger signal was detected at 24 h.
Presumably, by 24 h, the majority of the PLA2 had been secreted
into the lumen and was lost during washing of the gut sheets (Fig. 5).
Secreted PLA2 can be seen as a thick FITC signal between the epithelium
and the blood meal in 24-h transgenic gut sections (Fig. 6), and this
agrees with the Western data (Fig. 4).
Effect of PLA2 Expression on the Progression of Parasite
Development in the Mosquito and on Mosquito Vectorial Capacity--
To
investigate the effect of recombinant PLA2 expression on
P. berghei development, we fed both transgenic and
non-transgenic mosquitoes on the same infected mouse and counted
the number of oocysts that formed in each group of mosquitoes. As
indicated in Table I, infection
prevalence (column 3) and oocyst formation (column 4) were strongly
reduced in transgenic mosquitoes. In five independent experiments,
oocyst formation was inhibited from 77 to 99% (average inhibition
87%, column 5).
The effect of recombinant gene expression on the ability of mosquitoes
to transmit the parasite to uninfected animals (vectorial capacity) was
assessed by letting single infected mosquitoes feed on individual naive
mice and determining whether these mice became infected. As reported in
columns 2 and 3 of Table II, in every experiment, fewer transgenic than non-transgenic mosquitoes became infected, and the number of sporozoites in salivary glands of transgenic mosquitoes was correspondingly lower. More importantly, the
ability of transgenic mosquitoes to transmit the parasite to naive mice
was strongly inhibited. In three out of four experiments, transmission
of P. berghei parasites from infected transgenic mosquitoes
to naive mice was completely blocked, and in a fourth experiment, the
proportion of transgenic mosquitoes that transmitted the parasite was
much lower than in control wild type mosquitoes (Table II, column 4).
We considered the possibility that inhibition of parasite development
was a consequence of the fortuitous disruption of a mosquito gene upon
transposon insertion or of marker GFP expression. The following
considerations strongly argue against these possibilities: 1) the same
phenotype (inhibition of parasite development and transmission) was
observed in different mosquito lines, in which the transposon
integrated at different positions of the mosquito genome (Fig. 2 and
Tables I and II); 2) the same phenotype was observed when PLA2 was
provided exogenously to wild type mosquitoes (4); and 3) P. berghei developed equally well (oocyst and sporozoite numbers) in
transgenic mosquitoes that express only GFP and hygromycin
resistance gene (10) as in wild type
mosquitoes.2 The PLA2 protein
secreted in the midgut lumen of transgenic mosquitoes is most likely
responsible for inhibition of ookinete midgut invasion, as observed
with the exogenously administered protein (4).
The binding of phospholipases to their substrates, such as aggregated
phospholipids and membrane surfaces, is independent of their enzymatic
activity (19). Indeed, it has been shown that PLA2 inhibited
Plasmodium development even when its enzymatic activity was
inhibited (4), suggesting that PLA2 acts primarily via its binding to
exposed membrane lipids. Moreover, PLA2 had no effect on exflagellation
and zygote formation and did not affect normal ookinete motility on
glass slides, suggesting that this enzyme does not kill the parasite
(4). These considerations and the lipophylicity of the enzyme support
the hypothesis that PLA2 may be acting by interfering with the
interactions between Plasmodium and the midgut cell surface.
PLA2-expressing mosquitoes were normal in appearance and
lived as long as the non-transformed counterparts, although an abnormal
coloration of the blood meal was observed even 24 h after feeding
(bright red instead of dark brown). Egg laying may also be affected. A
detailed assessment of the fitness of transgenic as compared with wild
type mosquitoes is under way.
In summary, these experiments demonstrate that expression of the bee
PLA2 gene in the midgut of transgenic mosquitoes seriously compromises their ability to sustain Plasmodium development
and to transmit the parasite to other vertebrate hosts. Since this PLA2
also interferes with development and transmission of
Plasmodium falciparum in A. gambiae (4), we presume that PLA2 will be equally effective
in curtailing transmission in this most important parasite-vector
combination. Whereas reports of attempts to interfere with
Plasmodium transmission by expression of defensin (20) or
single chain antibodies (21) in A. aegypti have
appeared, their effectiveness in transgenic mosquitoes remains to be
demonstrated. Separate work from this laboratory indicates that
expression of SM1, a midgut and salivary gland binding peptide, also
effectively inhibits development and transmission of the parasite (6,
7). The availability of multiple targets to inhibit
Plasmodium development is crucial for future implementation
of the transgenic mosquito approach to reduce malaria transmission in
the field. This is because the Plasmodium genome is known
for its plasticity, and the possibility of the emergence of resistant
parasite strains needs to be avoided at all cost. Before one can
consider actual release assays, much work remains to be done. Issues
that need to be addressed include determination of wild mosquito
population structures, an evaluation of the ability of gene(s) to
spread through populations, considering the likelihood that the
parasites will develop resistance to the foreign effector gene product, and concerns about horizontal transfer. The experiments reported here
strongly suggest that genetic modification of mosquito vectorial capacity is feasible and represent a major step toward the goal of
containing the spread of malaria. However, for maximum effectiveness, we will have to rely on a multipronged approach that may include drugs,
insecticides, vaccines, and mosquito vectors expressing a combination
of effector genes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-32P]dCTP-labeled piggyBac
probe originating from the piggyBac vector left arm
(~0.8-kb SalI fragment from pBac
[3xP3-EGFP(AgCPPLA2)] (see Fig. 1, probe
a)).
80 °C. To determine the temporal profile of
PLA2 expression in transgenic mosquitoes, guts were
dissected at increasing time intervals following a blood meal. Total
RNA was extracted with TRI-Reagent (Molecular Research Center). Around
2.5 µg of total RNA was separated in each lane of a 1.5%
agarose-formaldehyde gel and blotted by capillary action to a nylon
membrane (Gene Screen). Hybridization was performed first with a
PLA2 probe (450 bp of coding region amplified by PCR; see
Fig. 1, probe b) and then with a mitochondrial rRNA probe
(13) as a loading control.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
View larger version (10K):
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Fig. 1.
Schematic diagram of the
AgCP-PLA2 gene inserted into the A.
stephensi germ line. The construct consists of
the AgCP promoter (open box) plus the 5'-UTR
(thick line), the AgCP signal sequence
(light stippled box), the coding sequence of bee PLA2
(dark stippled box), and the AgCP 3'-UTR
(thick continuous line on the right). The
bent arrow at the top localizes the transcription
initiation site. Dotted horizontal lines represent vector
sequences. The following restriction sites are indicated. A,
AscI; N, NotI; K,
KpnI; B, BamHI; F,
FseI; S, SacII; Bg,
BglII. This construct was introduced into the
AscI-FseI site of pBac
[3xP3-EGFPafm] plasmid for germ line transformation of
A. stephensi. a indicates the probe
used for Southern analysis (see "Experimental Procedures" and the
legend for Fig. 2). b indicates the probe used for Northern
analysis.
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[in a new window]
Fig. 2.
Verification of transgene integration by
Southern blot analysis. Genomic DNA from the transgenic
AgCP-PLA2 mosquito lines was digested with BglII,
blotted onto a nylon membrane, and hybridized with a 0.8-kb
piggyBac probe (Fig. 1, a). Different size
fragments were detected in the two lines that originated from each
family (A and B), thus demonstrating four different integration events.
The source of DNA is indicated at the top of each
lane.
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Fig. 3.
Time course of phospholipase A2 mRNA
accumulation after a blood meal. Total gut (or carcass) RNA was
extracted from transgenic AgCP-PLA2 BF4 mosquitoes dissected
at different times after a blood meal. The RNA was fractionated by gel
electrophoresis and blotted to a nylon membrane. The membrane was first
hybridized with a radioactive PLA2 probe and then with a mitochondrial
rRNA probe, used as a loading control. Numbers above the
lanes refer to the times after a blood meal when mosquitoes
were dissected. 0h, guts from sugar-fed mosquitoes;
G, guts; Carcass, non-gut tissues dissected at
4 h after a blood meal; NTG 4h, guts from
non-transformed mosquitoes dissected 4 h after a blood meal.
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Fig. 4.
Time course of recombinant PLA2 protein
expression. Guts from the non-transformed recipient
(NT) or from transgenic AgCP-PLA2 BF4 mosquitoes
were dissected at different times after a blood meal (indicated at the
top of the lanes). Protein equivalent of 0.4 gut
was analyzed on a Western blot with an anti-bee venom PLA2
antibody. The arrow on the right points to a
17-kDa band corresponding to PLA2 detected at 8, 16, and 24 h.
M, prestained molecular size marker (from top to
bottom: 190, 120, 85, 60, 50, 40, 25, 20, 15, 10 kDa). The
mobility of these markers is only approximately related to the
molecular sizes (indicated by ~ symbol).
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Fig. 5.
Immunofluorescence of midgut sheets.
Guts from the non-transformed recipient (NT) or from the
transgenic AgCP-PLA2 mosquitoes (TG) were
dissected at different times after a blood meal (indicated to the
left of the panels) and opened longitudinally to
make a sheet. The sheets were incubated with a rabbit antiserum to bee
venom phospholipase A2 followed by an anti-rabbit FITC-conjugated
antibody. The gut sheets were mounted on glass slides and examined by
differential interference contrast (left panels) or
fluorescence microscopy (right panels).
View larger version (80K):
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Fig. 6.
Immunofluorescence of midgut sections.
Fixed guts from non-transgenic recipient or from transgenic
AgCP-PLA2 BF4 mosquitoes, dissected at different times
after a blood meal (indicated to the left of the
panels), were sectioned and mounted onto glass slides. The
slides were incubated with a rabbit antiserum to bee venom
phospholipase A2 followed by a FITC-conjugated anti-rabbit antibody
(green signal). The nuclei of the midgut epithelial cells
(EC) are stained with DAPI (blue signal). The
ingested blood meal (BM) is also visible.
Effect of PLA2 expression on P. berghei oocyst formation
The vectorial capacity of transgenic mosquitoes is severely impaired
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ACKNOWLEDGEMENTS |
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We thank John Klapac for generously supplying two beehives containing young and emerging honeybees and also Greg Hundemer and Jason Snyder for excellent assistance with the handling of mosquitoes and mice. We are also grateful to members of our laboratory for helpful discussions and suggestions.
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FOOTNOTES |
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* This work was supported by grants from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF438408.
Corresponding author: Case Western Reserve University,
School of Medicine, Dept. of Genetics, 10900 Euclid Ave., Cleveland, OH
44106-4955. Tel.: 216-368-2791; Fax: 216-368-3432; E-mail: mxj3@po.cwru.edu.
Published, JBC Papers in Press, August 7, 2002, DOI 10.1074/jbc.M206647200
2 A. Crisanti, T. Nolan, and F. Catteruccia, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: PLA2, phospholipase A2; AgCP, A. gambiae carboxypeptidase; UTR, untranslated region; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; GFP, green fluorescent protein; EGFP, enhanced GFP; DAPI, 4',6-diamidino-2-phenylindole.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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