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    Schematic representation of the VgT2-containing transformation vector. The construct contains the right and left piggyBac-inverted repeat regions and associated DNA (pBacR and pBacL), the marker gene DsRed under control of the 3xP3 promoter, followed by the SV40 polyadenylation signal sequence, all derived from pBac[3xP3-DsRedafm].14 The An. gambiae VgT2 gene cis-regulatory elements (AgVgProm) are linked to the enhanced green florescent protein (EGFP) reporter gene and the bovine growth hormone (BGH) polyadenylation signal sequence.

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    Southern blot analyses of An. stephensi transformed lines, tVG1 and tVG2. (Top) Schematic representation of the transgene structure showing the size, relative location, and extent of the probes (probes A and B). (Bottom) Hybridization patterns of genomic DNA derived from the transgenic lines tVG1 and tVG2 digested with BamHI and the membrane hybridized with the EGFP probe (A). After exposure to film, the membrane was stripped and probed again with the piggyBac left-arm probe (B). Small arrowheads indicate discrete positive signals. Molecular mass markers are indicated on the left of image A. Abbreviations are as in Figure 1.

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    Expression of the reporter gene protein in transformed An. stephensi: (A) fluorescence in whole animals—(1) adult female non-blood-fed, non-transgenic (left) and transformed adult, female mosquito (right); the DsRed marker is detected easily in the eye of the transformed animal; (2) non-transformed adult female, 24 hPBM; (3) transformed adult female 24 hPBM; and (4) transformed adult female 48 hPBM; (B) immunoblot analysis of EGFP expression. NT, tVG1, and tVG2 indicate lanes containing extracts of non-transgenic and transgenic lines 24 hPBM. Positions of two molecular mass markers (in kDa) are indicated to the right of the image.

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    Analysis of EGFP mRNA accumulation in two separate lines of transgenic mosquitoes. RT-PCR techniques were performed on total RNA samples extracted from transgenic and non-transgenic mosquitoes. Three sets of primers were used in individual (left panels) or in multiplex reactions (right panels). EGFP, Vg, and rpS26 correspond to sets of primers specific for the enhanced green fluorescent protein, An. stephensi vitellogenin and the ribosomal protein S26 mRNAs, respectively. (Left panels) EGFP mRNA accumulation in larvae; pupae; transformed males, non-transformed (NT) adult females 24 hPBM; and adult transformed females of both strains, tVG1 and tVG2, as follows: non-blood-fed (NBF) and at 6, 24, 48, 72, and 96 hPBM. (Right panels) Tissue-specific expression of EGFP. RNA samples extracted 24 hPBM from fat-body tissues (FB), Malpighian tubules (MT), or ovaries (OV) of blood-fed transgenic females were used as templates for multiplex RT-PCR.

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    Accumulation of EGFP transcripts in blood-fed transgenic An. stephensi during three consecutive gonotrophic cycles. The EGFP mRNA accumulation pattern was determined after each of three consecutive bloodmeals taken at 96-h intervals. The levels of EGFP mRNA were determined semiquantitatively by RT-PCR at 24-h intervals. NBF, non-blood-fed females; 24, 48, 72, and 96 indicate hPBM in each of the three gonotrophic cycles. All reactions were performed with 0.5 μg of DNase I-treated total RNA as template.

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    Alignment of the putative cis-acting regulatory elements of Anopheles vitellogenin-encoding gene promoters. Four sequences corresponding to vitellogenin-encoding genes, AngaVg1 and AngaVg2 (VgT1 and VgT2, respectively; GenBank accession number AF281078) for An. gambiae and AnstVg1 and AnstVg2 (GenBank accession number DQ442990) for An. stephensi, were aligned to evaluate sequence identities and similarities. Asterisks indicate identity among all four sequences. The transcription initiation sites are boxed and designated +1 in all sequences. An arthropod consensus transcription initiation sequence includes the initiator nucleotides (brown and shaded). The ATG initiation codon also is boxed. GATA and GATA-like sequences are shown in green. The putative C/EBP binding sites are orange, and EcRE sites are indicated in blue.

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THE ANOPHELES GAMBIAE VITELLOGENIN GENE (VGT2) PROMOTER DIRECTS PERSISTENT ACCUMULATION OF A REPORTER GENE PRODUCT IN TRANSGENIC ANOPHELES STEPHENSI FOLLOWING MULTIPLE BLOODMEALS

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  • 1 Department of Parasitology, School of Public Health and Tropical Medicine, Southern Medical University, Guang Zhou, GD 510515, P.R. China; Department of Molecular Biology & Biochemistry, University of California, Irvine, CA 92697-3900; Department of Microbiology & Molecular Genetics, University of California, CA 92697-4025

Mosquitoes made resistant to pathogens through genetic engineering are proposed as a basis for developing a strategy to control disease transmission. Transgenic approaches that introduce exogenous antipathogen effector genes into mosquito genomes require cis-acting regulatory DNA to control tissue-, stage-, and sex-specific transgene expression. We show that control sequences derived from a vitellogenin-encoding gene of Anopheles gambiae, a major vector in sub-Saharan Africa, can direct expression of an exogenous gene in a tissue-, stage-, and sex-specific manner in Anopheles stephensi, a vector of urban malaria in southern Asia. Specific reporter gene expression was observed in fat-body tissues of transgenic blood-fed females, but not in transgenic males or non-blood-fed transgenic females. Multiple bloodmeals resulted in the continuous presence of reporter gene transcripts for at least 12 days. The persistent expression makes the heterologous promoter a good candidate for controlling transcription of engineered antipathogen effector genes in this important malaria vector.

INTRODUCTION

One approach to genetic control of transmission of the parasites that cause human malaria is based on deploying transgenic mosquitoes expressing antiparasitic effector genes that disable the pathogens.1,2 An essential step in engineering such mosquitoes with reduced vector competence is the identification of suitable promoters to drive the expression of the effector genes.3,4 The search for appropriate promoters resulted in the identification and characterization of DNA sequences containing regulatory elements capable of controlling the temporal and spatial expression of exogenous genes. For example, a carboxypeptidase gene promoter included in a transgene limits the expression of its product to the midgut of genetically modified mosquitoes5,6 and the promoter of a gene encoding an anopheline antiplatelet protein directs transgene expression to the salivary glands.7 The list of functional mosquito control DNA sequences also includes those that drive the expression of genes in the fat-body tissues,8 flight muscles,9 and gonads.10 The regulatory elements of vitellogenin-encoding genes (Vg) have been studied extensively based primarily on the strong induction of transcription following a bloodmeal and restricted expression in the fat body of adult females (vitellogenins are the major yolk protein precursor in mosquitoes). The peak accumulation of Vg-encoded mRNA corresponds approximately to the time at which the human malaria parasites are ookinetes invading the midgut. Although it is possible that a fat-body–expressed effector molecule could traverse the basal membrane of the midgut epithelium and inactivate the penetrating ookinete or disrupt the developing oocyst, Vg control sequences should be most useful for targeting an effector molecule to the sporozoites in the hemolymph. Although the initial temporal expression of endogenous Vg genes, 24–48 h after a bloodmeal, does not overlap the appearance of sporozoites in the hemolymph, which occurs much later (8–16 days, depending on species and temperature) after an infective bloodmeal,11 studies have shown that some anophelines, including Anopheles gambiae and Anopheles stephensi, take multiple blood-meals within each gonotrophic cycle.4,12 This repetitive feeding could induce sufficient transcription from a Vg promoter-driven transgene, resulting in an effector molecule being present in the hemolymph long enough to disrupt sporozoites. Considering the potential of Vg cis-regulatory elements for the development of mosquito transgenesis-related technologies, we tested and demonstrated that the cis-acting sequences of the An. gambiae VgT2 gene (GenBank accession number AF281078) can drive persistent sex- and tissue-specific expression of a reporter gene in another malaria vector species, An. stephensi, after multiple bloodmeals.

MATERIALS AND METHODS

Mosquitoes.

An. stephensi larvae (NIH strain, gift of M. Jacobs-Lorena, Johns Hopkins University) were fed on finely ground fish food (Tetramin, Doctors, Foster and Smith, Rhinelander, WI) mixed 1:1 with yeast powder. Adults were maintained at 25°C, 75–85% relative humidity, and 18 h/6 h light/dark cycles, and were fed ad libitum on raisins and water. Adult females were fed 3–4 days after eclosion on anesthetized mice when a bloodmeal was required. For multiple bloodmeals, mosquitoes were allowed to feed on mice for ≈30 minutes, and those that had fed to repletion were separated from the cohort and transferred to a new cage. A second bloodmeal was given after 96 h, and the females that fed a second time were transferred to a new cage. Similarly, a third bloodmeal was provided 96 h later and the blood-fed females were separated. From these blood-fed groups, 3–4 females were collected at 24, 48, 72, and 96 h post-bloodmeal (hPBM) for analysis of gene expression by one-step RT-PCR. During the entire procedure, a cup filled with water was placed in all the cages to facilitate oviposition.

Cloning of the putative cis-regulatory region of the VgT2 gene from An. gambiae and construction of the transformation vector.

On the basis of the published genomic sequence of An. gambiae VgT2 (Genbank accession number AF281078), a pair of primers, VgPromforECO (5′ CG-GAATCTTGGTCCGCAATAATGAAAGTT 3′) and Vg-PromrevBAM (5′ CGGGATCCGGTTCGGTTGTTCG-CAGTTG 3′) were designed and used in gene amplification protocols with An. gambiae genomic DNA as template. The amplified fragment, 1.7 kb in length, contains the putative regulatory region of VgT2 and was cloned into the pGlow TOPO vector (Invitrogen, Carlsbad, CA). After being sequenced to verify orientation and reading frame integrity, the DNA fragment containing the Vg promoter, EGFP-coding sequence, and bovine growth hormone (BGH) polyadenylation region were subcloned into the shuttle vector, pSLFa,13 and then cloned into the AscI site of the transformation vector pBac[3xP3-DsRedafm]14 to generate the plas-mid pBac[3xP3DsRed-AgVgT2-GFP] (Figure 1). The transformation construct was designed without a signal peptide to facilitate the analysis of spatial patterns of expression. The orientation and junctions of the cloned DNA fragments were verified by sequencing.

Embryo microinjection.

Microinjection of An. stephensi embryos was performed as described.15 Embryos were micro-injected with the construct pBac[3xP3DsRed-AgVgT2-GFP] plus the helper plasmid, phsp-pBac, and allowed to develop into adults. Surviving injected animals (G0) were mated as male and female pools consisting of 3–5 G0 males out-crossed to 45–100 wild-type females or 10 G0 females out-crossed to 10 wild-type males. Sixty-seven matings were performed, and G1 larvae were screened under a fluorescent microscope. Transgenic families were identified, and the DsRed-positive individuals were intercrossed for all subsequent generations for propagation and further analyses of the transgenic lines.

Southern blot analyses.

Genomic DNA was extracted from adult mosquitoes 5 days after eclosion, digested with BamHI, 14 μg resolved on a 0.8% agarose gel, and the gel-blotted on to Zeta-probe GT Genomic Tested Blotting Membranes (Bio-Rad, Hercules, CA) using standard protocols.16 Two probes were used for hybridization with DNA extracted from transgenic mosquitoes. One consisted of the EGFP open-reading frame (ORF) 723 bp in length amplified from the plasmid pBac[3xP3DsRed-AgVgT2-GFP] using primers GFP for (5′-ATGGCTAGCAAAGGAGAAGAAC-3′) and GFP rev (5′-ATTTGTAGAGCTCATCCATGCC-3′), and the other was the DNA sequence corresponding to the piggyBac left arm (≈0.9 kb) generated by AscI and EcoRI double-digestion of pBac[3xP3-DsRedafm]. The probes were 32P-labeled using a random primer labeling kit (Amersham-Pharmacia, Piscataway, NJ). Membrane hybridization was carried out under high-stringency conditions at 65°C overnight.17 After hybridization, the membrane was washed twice at 65°C for 15 min with 2× SSC/0.1%SDS and twice with 0.1× SSC/0.1%SDS at 65°C for 15 min. Kodak Biomax MR film (Kodak, Rochester, NY) was exposed to membranes with an intensifying screen for 24 h at −80°C.

Gene amplification of mRNA (RT-PCR).

Total RNA was isolated from 5- to 7-day-old sugar-fed males and non-blood-fed and blood-fed females at several time points PBM. RNA samples also were extracted from immature stages and dissected tissues. One-step RT-PCR of the transcription products of an endogenous An. stephensi vitellogenin-encoding gene, AsVg1 (GenBank accession number DQ442990), was performed using specific primers, AsVg1for (5′-CAACATCATGTCCAAGTCGGAGGTGA-3′) and AsVg1rev (5′-CTTGAAGCTTTCGTGCTCTTCCTCCG-3′), that amplify a product 450 bp in length. For functional analysis of the An. gambiae vitellogenin cis-regulatory sequence in transgenic lines, one-step RT-PCR was performed on total RNA using EGFP gene-specific primers that amplify a product 723 bp in length. As an internal control, the transcription product of the putative An. stephensi ribosomal protein S26 gene (GenBank accession number CB367652)18 was amplified using primers Asrib26Sfor (5′-AATCCTTC-CCGAAGGACATGAACCG-3′) and Asrib26Srev (5′-TACGAAACAAATCCCATCCTAATCGAAGC-3′) to yield a 196-bp fragment.

SDS-PAGE and immunoblots.

One adult mosquito was homogenized 24 hPBM in 100 μL of water containing 1× concentration of Complete Mini Protease Inhibitor Cocktail (Roche, Indianapolis, IN) plus 1 mg/mL Pefabloc SC (Roche) and was then mixed with 100 μL of SDS-sample buffer.19 The samples were treated at 95°C for 5 min and centrifuged at 13,500 rpm for 5 min. Aliquots of 20 μL of supernatant were loaded into the wells of a 12% polyacrylamide gel, and the proteins resolved by electrophoresis. After electrophoresis, proteins either were stained with Coomassie Blue R or transferred to Hybond-P PVDF membranes using standard protocols.16 Rabbit polyclonal IgG anti GFP (FL) (Santa Cruz Biotechnology, Santa Cruz, CA) was used as the primary antibody (1:4,000 dilution) and Goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology) (1:100,000 dilution) was the secondary reagent. The substrate 3-chloro-1-naphthol (Sigma, St. Louis, MO) was used for visualization of reactive bands.16

RESULTS

Mosquito transformation.

A total of 1,062 An. stephensi embryos were microinjected with the transformation construct and helper plasmid; of these, 224 larvae hatched and 141 adults emerged. Two transgenic families were identified (tVG1 and tVG2) by expression of the DsRed marker in the eyes of the insects. Integration of the transgenes into the genome was demonstrated by Southern blot analyses (Figure 2). Complete digestion of the genomic DNA with BamHI results in the generation of a diagnostic fragment of ≈540 bp that hybridizes with the EGFP probe. The probe also hybridizes weakly with higher molecular weight DNA fragments that contain 196 nucleotides (nt) of the EGFP 3′-end in addition to the piggyBac left arm and mosquito genomic DNA. To validate the high molecular weight signals observed with the EGFP probe, the blots were stripped and reprobed with the piggyBac left arm. The additional high molecular weight DNA fragments, visible after hybridization with both probes, are consistent with multiple (at least nine) independent insertions of the transgene in tVG1 and three in tVG2.

Functional analysis of the An. gambiae vitellogenin putative cis-regulatory sequence.

Expression of the EGFP ORF in the transgenic mosquitoes was analyzed by direct visualization of green fluorescence and RT-PCR. Green fluorescence was observed in both transgenic lines, and there was an observable increase of fluorescence after a bloodmeal (Figure 3). Non-transgenic An. stephensi adults and immature stages display autofluorescence when observed under the EGFP filter and excitation light used in our studies. Therefore, to corroborate the fluorescence observations, we determined the expression of EGFP by immunoblots and RT-PCR, using EGFP-specific antibodies and gene-specific primers, respectively. A species of ≈30 kDa reacts with anti-EGFP antibodies and is present in transgenic but not non-transgenic mosquitoes (Figure 3). This is consistent with the appropriate synthesis of the reporter molecule.

Gene amplification RT-PCR revealed that EGFP mRNA accumulates only in transgenic females after bloodmeal (Figure 4). No EGFP mRNA was detected in transgenic larvae, pupae, adult males, or non-blood-fed females. Furthermore, no amplification products were obtained from non-transgenic mosquitoes. RT-PCR also was performed with RNA extracted from dissected tissues of blood-fed females. Accumulation of EGFP mRNA was observed exclusively in the fat-body tissues of the mosquitoes. These results indicate that the An. gambiae VgT2 cis-regulatory sequences contain elements capable of controlling tissue- and sex-specific expression in An. stephensi. Importantly, the transgene did not follow a temporal-expression pattern identical to the endogenous vitellogenin genes. Although the endogenous vitellogenin gene is expressed almost exclusively at 24 and 48 hPBM, EGFP mRNA could be detected up to 96 hPBM in both transgenic lines.

Multiple bloodmeals and transgene expression.

The effect of consecutive bloodmeals and gonotrophic cycles on the expression of the transgene also was determined. Mosquitoes were fed on blood, allowed to lay eggs (eggs are laid 72–96 hPBM in our laboratory conditions), and blood-fed again. RT-PCR experiments demonstrated that EGFP mRNA is induced after each bloodmeal in both lines and is present continuously in tVG1 transgenic females up to the end of the third gonotrophic cycle (Figure 5). Each bloodmeal is followed by an immediate (24 hPBM) accumulation of EGFP mRNA and a decline in its abundance during the next 3 days. EGFP mRNA was present at detectable levels in all samples analyzed, up to 12 days after the first bloodmeal. The decrease in signal corresponding to the EGFP mRNA that is observed from the first to the third gonotrophic cycle is consistent with reports that older females produce fewer eggs in each subsequent cycle.20

Putative cis-regulatory elements in anopheline vitellogenin promoters.

The regulatory activity of the An. gambiae VgT2 promoter in An. stephensi supports the hypotheses that the two species may share similar cis-acting regulatory DNA for their respective vitellogenin-encoding genes. Alignment of the DNA sequences at the 5′-ends of four vitellogenin genes—two from An. gambiae and two from An. stephensi—shows that the DNA proximal to the expressed sequence is highly-conserved in a region extending ≈250 nt to the 5′-end of the transcription start site (Figure 6). Increasing divergence is observed with distance from the start site. A typical arthropod transcription initiator sequence21 is located 27 nt to the 3′-end of a TATA motif in all four sequences. Furthermore, the translation initiation sites (ATG) also are conserved in their relative locations (+56 to +58 nt). In addition to the basal promoter elements, putative transcription factor binding sites (EcRE, GATA, C/EBP) similar to those required for correct tissue- and stage-specific expression of the Aedes aegypti Vg1 gene22 are conserved both in sequence and relative position among the anopheline genes.

DISCUSSION

Genetics-based technologies focused on limiting the size of mosquito vector populations (population reduction) or altering the populations so that they are resistant to pathogens (population modification/replacement) are expected to provide supplemental and alternative means of controlling transmission of the etiological agents of malaria, dengue, and other diseases.23 Crucial to these proposed strategies is the ability to genetically manipulate the vector species, introgress new genes at high rates into target populations, and identify anti-pathogen effector molecules and DNA regulatory elements to drive the expression of these genes in the appropriate tissue and developmental stage of the mosquito.23,24

The objective of this study was to evaluate the functionality of the An. gambiae vitellogenin promoter in transgenic An. stephensi mosquitoes to control the expression of an anti-malarial gene in the fat-body tissues of the vector insect and to target the parasites in the mosquito hemolymph. Anopheles gambiae and An. stephensi are closely related species belonging to the same subgenus (Cellia),25 and gene-regulatory mechanisms are likely conserved between these two taxa. For example, the An. gambiae adult peritrophic matrix protein 1 gene (AgAper1) regulatory elements contained in a 2.5-kb putative promoter were sufficient to direct the accumulation of phospholipase A2 (PLA2) in transgenic An. stephensi.26 Genomic fragments located to the 5′-end of the An. gambiae female salivary gland-specific genes AgApy (≈800 bp) and D7r4 (≈1.0 kb) directed expression of reporter genes in the salivary glands of transgenic An. stephensi.27 However, in this case, expression levels were low, and tissue-and sex-specific expression of the transgenes were not maintained in the transgenic insects.

Conservation of regulatory elements also has been demonstrated between anopheline and culicine mosquitoes. The An. gambiae carboxypeptidase promoter (3.4 kb) is capable of controlling expression of a reporter gene in the gut cells of An. stephensi and Ae. aegypti.5,28 Recently, the reciprocal was demonstrated when the Ae. aegypti carboxypeptidase promoter (≈1.2 kb) was used to control expression of transgenes in An. gambiae.6 Expression of the reporter genes under the control of mosquito promoters also was observed in transgenic Drosophila melanogaster, indicating some conservation of control elements among the fruit fly and mosquitoes.27,29,30

The promoters of genes expressed in the salivary glands are good candidates to direct the expression of anti-pathogen genes targeting malaria sporozoites and those from genes expressed in the gut cells are ideal to target the gametocytes and ookinetes within the mosquitoes. A third group composed of genes expressed in the fat-body tissues is of interest as a fat-body–expressed effector molecule could traverse the basal membrane of the midgut epithelium and inactivate the penetrating ookinetes or disrupt the developing oocysts. However, the most accessible target of a fat-body–expressed antipathogen molecule would be the sporozoites in the hemolymph.

Among the fat-body–expressed genes, those encoding vitellogenins have been studied extensively resulting in a considerable understanding of their physiology, gene regulation, cis-acting promoter elements, and transcription factors.8,31 Kokoza and others32,33 and Shin and others34 used a promoter DNA fragment (2.1-kb) from the Ae. aegypti Vg1 gene to drive expression of antimicrobial effector proteins in transgenic adult females.8,3134 Over-expression in the fat body and secretion into the hemolymph of cecropin inhibited partially oocyst growth of the avian malaria parasite, Plasmodium gallinaceum.34 The Ae. aegypti Vg1 gene promoter used in those studies was competent to provide bloodmeal-induced expression of transgenes in female mosquitoes. However, low-level expression also was seen in transgenic males.32 It is likely that regulatory elements required for a complete repression of the Vg1 gene expression in males were not present in the 2.1-kb 5′-end of the gene used in that work. Additional work with Ae. aegypti mosquitoes identified regulatory elements such as GATA motifs, distributed from −111 to −1902 bp to the 5′-end of the VgA1 gene, and repressor proteins presumably are displaced from these motifs for activator binding. Other regulatory elements in addition to the 2.1-kb sequence used likely are located further to the 5′-end.22 Recently, Nirmala and others8 determined that the 850 nucleotides immediately adjacent to the 5′-end and the 3′UTR of An. stephensi AsVg1 gene are sufficient to direct sex-, stage-, and tissue-specific expression of a reporter gene, and this is consistent with the interpretation that the cis-regulatory sequences in anophelines may be localized in a more compact DNA sequence than those previously studied in Ae. aegypti.

Two An. gambiae vitellogenin genes have been characterized and the corresponding 5′-end putative cis-regulatory sequences are available in GenBank (accession number AF281078). Here we demonstrate that the DNA sequence containing the An. gambiae VgT2 gene 5′-end control DNA can drive tissue- and sex-specific expression of a reporter gene in An. stephensi. However, the expression of the transgene did not follow a pattern identical to that observed for the endogenous vitellogenin genes of either An. gambiae or the An. stephensi. In these mosquito species, vitellogenin genes are expressed after a bloodmeal, with the mRNA accumulation peaking at 24–48 hPBM.8,3538 The EGFP mRNA in both transgenic lines was expressed only in the fat-body tissues of blood fed-females, however its concentration did not decline after 24–48 hPBM as it occur with the endogenous Vg genes. EGFP mRNAs are present even at 72–96 hPBM indicating that either the promoter is not repressed, resulting in continuous transcription, or that the transgene mRNA has a longer turnover period.

When the An. stephensi Vg1 promoter was tested to drive expression of a reporter molecule in the homologous species, expression of the transgene followed a pattern identical to that of the endogenous Vg gene, peaking at 24–48 hPBM.8 This expression profile compared with that seen with the heterologous VgT2 pattern highlights the possibility that transcription factors (TF) responsible for the repression of Vg gene expression at the end of each gonotrophic cycle, differ in these two species, and that the An. stephensi regulatory factors do not bind adequately to the An. gambiae Vg promoter. Experiments aiming to identify TF binding sites in these An. gambiae and An. stephensi promoters will be necessary to test this hypothesis. Alternatively, the stability of the transgene mRNA may differ from that of the endogenous Vg genes. The 3′UTR sequence of the transgene used in the experiments described here is derived from a mammalian gene (BGH) instead of the mosquito Vg gene, and it has already been established that the noncoding 3′- and 5′-ends of mRNAs molecules participate in essential processes that regulate both translation and RNA degradation.39

Independent of the mechanisms that resulted in the prolonged presence of EGFP mRNA in the transgenic mosquitoes, this feature is desirable for the purpose of expressing an anti-malarial molecule in An. stephensi. An extended presence of the transgene product would increase the chances of the effector molecule being in the hemolymph at a time when the parasites are migrating from the gut to the salivary glands of the mosquitoes. With multiple bloodmeals in each gonotrophic cycle,4,12,40 this repetitive feeding could induce sufficient transcription of a Vg promoter-driven transgene, resulting in an effector molecule being present in the hemolymph at an effective concentration and long enough to disrupt sporozoites.

Figure 1.
Figure 1.

Schematic representation of the VgT2-containing transformation vector. The construct contains the right and left piggyBac-inverted repeat regions and associated DNA (pBacR and pBacL), the marker gene DsRed under control of the 3xP3 promoter, followed by the SV40 polyadenylation signal sequence, all derived from pBac[3xP3-DsRedafm].14 The An. gambiae VgT2 gene cis-regulatory elements (AgVgProm) are linked to the enhanced green florescent protein (EGFP) reporter gene and the bovine growth hormone (BGH) polyadenylation signal sequence.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 76, 6; 10.4269/ajtmh.2007.76.1118

Figure 2.
Figure 2.

Southern blot analyses of An. stephensi transformed lines, tVG1 and tVG2. (Top) Schematic representation of the transgene structure showing the size, relative location, and extent of the probes (probes A and B). (Bottom) Hybridization patterns of genomic DNA derived from the transgenic lines tVG1 and tVG2 digested with BamHI and the membrane hybridized with the EGFP probe (A). After exposure to film, the membrane was stripped and probed again with the piggyBac left-arm probe (B). Small arrowheads indicate discrete positive signals. Molecular mass markers are indicated on the left of image A. Abbreviations are as in Figure 1.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 76, 6; 10.4269/ajtmh.2007.76.1118

Figure 3.
Figure 3.

Expression of the reporter gene protein in transformed An. stephensi: (A) fluorescence in whole animals—(1) adult female non-blood-fed, non-transgenic (left) and transformed adult, female mosquito (right); the DsRed marker is detected easily in the eye of the transformed animal; (2) non-transformed adult female, 24 hPBM; (3) transformed adult female 24 hPBM; and (4) transformed adult female 48 hPBM; (B) immunoblot analysis of EGFP expression. NT, tVG1, and tVG2 indicate lanes containing extracts of non-transgenic and transgenic lines 24 hPBM. Positions of two molecular mass markers (in kDa) are indicated to the right of the image.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 76, 6; 10.4269/ajtmh.2007.76.1118

Figure 4.
Figure 4.

Analysis of EGFP mRNA accumulation in two separate lines of transgenic mosquitoes. RT-PCR techniques were performed on total RNA samples extracted from transgenic and non-transgenic mosquitoes. Three sets of primers were used in individual (left panels) or in multiplex reactions (right panels). EGFP, Vg, and rpS26 correspond to sets of primers specific for the enhanced green fluorescent protein, An. stephensi vitellogenin and the ribosomal protein S26 mRNAs, respectively. (Left panels) EGFP mRNA accumulation in larvae; pupae; transformed males, non-transformed (NT) adult females 24 hPBM; and adult transformed females of both strains, tVG1 and tVG2, as follows: non-blood-fed (NBF) and at 6, 24, 48, 72, and 96 hPBM. (Right panels) Tissue-specific expression of EGFP. RNA samples extracted 24 hPBM from fat-body tissues (FB), Malpighian tubules (MT), or ovaries (OV) of blood-fed transgenic females were used as templates for multiplex RT-PCR.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 76, 6; 10.4269/ajtmh.2007.76.1118

Figure 5.
Figure 5.

Accumulation of EGFP transcripts in blood-fed transgenic An. stephensi during three consecutive gonotrophic cycles. The EGFP mRNA accumulation pattern was determined after each of three consecutive bloodmeals taken at 96-h intervals. The levels of EGFP mRNA were determined semiquantitatively by RT-PCR at 24-h intervals. NBF, non-blood-fed females; 24, 48, 72, and 96 indicate hPBM in each of the three gonotrophic cycles. All reactions were performed with 0.5 μg of DNase I-treated total RNA as template.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 76, 6; 10.4269/ajtmh.2007.76.1118

Figure 6.
Figure 6.

Alignment of the putative cis-acting regulatory elements of Anopheles vitellogenin-encoding gene promoters. Four sequences corresponding to vitellogenin-encoding genes, AngaVg1 and AngaVg2 (VgT1 and VgT2, respectively; GenBank accession number AF281078) for An. gambiae and AnstVg1 and AnstVg2 (GenBank accession number DQ442990) for An. stephensi, were aligned to evaluate sequence identities and similarities. Asterisks indicate identity among all four sequences. The transcription initiation sites are boxed and designated +1 in all sequences. An arthropod consensus transcription initiation sequence includes the initiator nucleotides (brown and shaded). The ATG initiation codon also is boxed. GATA and GATA-like sequences are shown in green. The putative C/EBP binding sites are orange, and EcRE sites are indicated in blue.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 76, 6; 10.4269/ajtmh.2007.76.1118

*

Address correspondence to Anthony James, Department of Molecular Biology & Biochemistry, 3205 McGaugh Hall, University of California, Irvine, CA 92697-3900 Irvine, CA. E-mail: aajames@uci.edu

Authors’ addresses: Xiao-Guang Chen, Department of Parasitology, School of Public Health and Tropical Medicine, Southern Medical University, Guang Zhou, GD 510515, P.R. China. Osvaldo Marinotti, Lucia Whitman, and Nijole Jasinskiene, Department of Molecular Biology & Biochemistry, University of California, Irvine, CA 92697-3900. Anthony A. James, Department of Microbiology & Molecular Genetics, University of California, CA 92697-4025 and Department of Molecular Biology & Biochemistry, University of California, Irvine, CA 92697-3900.

Acknowledgments: The authors thank Lynn Olson for help in preparing the manuscript.

Financial support: This study was supported by grants from The National Institutes of Health (AI29746) to A.A.J. and by a State Scholarship Fund from China Scholarship Council and National Natural Science Foundation of China (no. 30271162) to X.-G.C.

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Author Notes

Reprint requests: Anthony A. James, Department of Molecular Biology & Biochemistry, 3205 McGaugh Hall, University of California, Irvine CA 92697-3900, Telephone: +1 (949) 824-5930, Fax: +1 (949) 824-2814, E-mail: aajames@uci.edu.
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