FLP-mediated recombination in the vector mosquito, Aedes aegypti.

The activity of a yeast recombinase, FLP, on specific target DNA sequences, FRT, has been demonstrated in embryos of the vector mosquito, Aedes aegypti. In a series of experiments, plasmids containing the FLP recombinase under control of a heterologous heat-shock gene promoter were co-injected with target plasmids containing FRT sites into preblastoderm stage mosquito embryos. FLP-mediated recombination was detected between (i) tandem repeats of FRT sites leading to the excision of specific DNA sequences and (ii) FRT sites located on separate plasmids resulting in the formation of heterodimeric or higher order multimeric plasmids. In addition to FRT sites originally isolated from the yeast 2 microns plasmid, a number of synthetic FRT sites were also used. The synthetic sites were fully functional as target sites for recombination and gave results similar to those derived from the yeast 2 microns plasmid. This successful demonstration of yeast FLP recombinase activity in the mosquito embryo suggests a possible future application of this system in establishing transformed lines of mosquitoes for use in vector control strategies and basic studies.     

Organization of a cloned repetitive DNA fragment in mosquito genomes (Diptera: Culicidae).

The structure and genomic organization of a cloned 5.2-kb repetitive DNA fragment, H-85, isolated from the Aedes albopictus genome have been examined. In situ hybridization of the 3H-labeled H-85 DNA to the meiotic and mitotic chromosome preparations of Ae. albopictus shows that the sequences homologous to H-85 DNA are dispersed throughout the length of all three pairs of chromosomes. A similar pattern of in situ hybridization appears in Aedes seatoi, Aedes flavopictus, and Aedes aegypti. The study shows that the arrangement of sequences in the cloned 5.2-kb fragment is rare in the Ae. albopictus genome. Dot-blot hybridization reveals that the sequences homologous to H-85 DNA are present in 12 species of mosquitoes examined, belonging to six genera in subfamilies Culicinae ad Anophelinae. The H-85 sequences are also present in the genome of Mochlonyx velutinus of the nematocerous family Chaoboridae, earlier proposed as the ancestor of the mosquito family Culicidae. Although the sequences homologous to H-85 DNA are present in different species of mosquitoes, they have diverged in their structure and organization. The cloned 5.2-kb fragment is composed of elements of different and independently evolving repetitive DNA families.     

The Aedes aegypti genome: complexity and organization.

We describe the use of DNA reassociation kinetics to determine the total genome size and complexity together with the individual complexity and copy number of the single copy, middle repetitive and highly repeated DNA fractions of cell line and larval DNA from the mosquito, Aedes aegypti. The genome of Ae. aegypti is both large and complex, being one third the size of the human genome, and exhibits a short period interspersed repeat pattern. The implications of patterns of sequence arrangement and genome complexities for experiments aimed at isolating specific classes of DNA sequences, such as mobile genetic elements, are discussed.     

High efficiency, site-specific excision of a marker gene by the phage P1 cre-loxP system in the yellow fever mosquito, Aedes aegypti

The excision of specific DNA sequences from integrated transgenes in insects permits the dissection in situ of structural elements that may be important in controlling gene expression. Furthermore, manipulation of potential control elements in the context of a single integration site mitigates against insertion site influences of the surrounding genome. The cre-loxP site-specific recombination system has been used successfully to remove a marker gene from transgenic yellow fever mosquitoes, Aedes aegypti. A total of 33.3% of all fertile families resulting from excision protocols showed evidence of cre-loxP-mediated site-specific excision. Excision frequencies were as high as 99.4% within individual families. The cre recombinase was shown to precisely recognize loxP sites in the mosquito genome and catalyze excision. Similar experiments with the FLP/FRT site-specific recombination system failed to demonstrate excision of the marker gene from the mosquito chromosomes.     

Microsatellite isolation and linkage group identification in the yellow fever mosquito Aedes aegypti

Microsatellites have proved to be very useful as genetic markers, as they seem to be ubiquitous and randomly distributed throughout most eukaryote genomes. However, our laboratories and others have determined that this paradigm does not necessarily apply to the yellow fever mosquito Aedes aegypti. We report the isolation and identification of microsatellite sequences from multiple genomic libraries for A. aegypti. We identified 6 single-copy simple microsatellites from 3 plasmid libraries enriched for (GA)(n), (AAT)(n), and (TAGA)(n) motifs from A. aegypti. In addition, we identified 5 single-copy microsatellites from an A. aegypti cosmid library. Genetic map positions were determined for 8 microsatellite loci. These markers greatly increase the number of microsatellite markers available for A. aegypti and provide additional tools for studying genetic variability of mosquito populations. Additionally, most A. aegypti microsatellites are closely associated with repetitive elements that likely accounts for the limited success in developing an extensive panel of microsatellite marker loci.     

Agrobacterium-mediated sorghum transformation

Agrobacterium tumefaciens was used to genetically transform sorghum. Immature embryos of a public (P898012) and a commercial line (PHI391) of sorghum were used as the target explants. The Agrobacterium strain used was LBA4404 carrying a `Super-binary' vector with a bar gene as a selectable marker for herbicide resistance in the plant cells. A series of parameter tests was used to establish a baseline for conditions to be used in stable transformation experiments. A number of different transformation conditions were tested and a total of 131 stably transformed events were produced from 6175 embryos in these two sorghum lines. Statistical analysis showed that the source of the embryos had a very significant impact on transformation efficiency, with field-grown embryos producing a higher transformation frequency than greenhouse-grown embryos. Southern blot analysis of DNA from leaf tissues of T₀ plants confirmed the integration of the T-DNA into the sorghum genome. Mendelian segregation in the T₁ generation was confirmed by herbicide resistance screening. This is the first report of successful use of Agrobacterium for production of stably transformed sorghum plants. The Agrobacterium method we used yields a higher frequency of stable transformation that other methods reported previously.     

吸血诱导的埃及伊蚊海口株后期胰蛋白酶基因测序及与美国株同源性比较

应用逆转录-聚合酶链式反应（RT-PCR）技术从吸血后24 h埃及伊蚊海口株总RNA中扩增出了后期胰蛋白酶编码区cDNA序列。采用自动DNA分析仪进行序列分析,并与已知埃及伊蚊美国株后期胰蛋白酶基因及推导的氨基酸序列进行了同源性比较。结果表明：埃及伊蚊海口株后期胰蛋白酶基因序列与美国株同源性达98%,有11个碱基发生变异;氨基酸同源性达99%,仅有3个氨基酸发生变异,但与催化位点密切相关的氨基酸及N末端氨基酸序列完全一致。以上结果显示,埃及伊蚊胰蛋白酶不同地理株间存在微小的差异。     

Self-Fertile Transgenic Wheat Plants Expressing Herbicide Bromoxynil Resistance

Genetic manipulation of wheat (Triticum aestivum L. ) by biotechnological approaches is currently limited by a lack of efficient and reliable transformation method. The authors report a reproducible protocol for rapid production of transgenic wheat via microprojectile bombardment. The experiment was carried out by using the immature embryo excised from caryopsis 14 to 18 days postanthesis and the plant expression plasmid carrying a CaMV 35S-controlled bxn gene, for resistance to herbicide bromoxynil and a selectahle marker gene NPT I. After bombarding the precultured immature embryos isolated from 13 wheat varieties with plasmid DNA-coated tungsten particle, these embryos were transferred on MS medium containing 10 mg/L geneticin G418 sulphate to select and regenerate transformants step by step. As a result, 16 transformed plants were obtained from a total of 849 bombarded embryos. The characterization of these plants by inoculation with herbicide bromoxynil and Southern analysis with bxn gene as a probe showed that 4 of the self-fertile transformed plants contained the target gene and presented herbicide resistance. In several independent transformation experiments, the fastest one took only 6 months from embryo excision to characterization of regenerated plants. Therefore, this procedure is a rapid and efficient technique for delivering foreign DNA into wheat.     

Tn5 as an insect gene vector

The purpose of this study was to explore alternatives to insect-derived transposable elements as insect gene vectors with the intention of improving existing insect transgenesis methods. The mobility properties of the bacterial transposon, Tn5, were tested in mosquitoes using a transient transposable element mobility assay and by attempting to create transgenic insects. Tn5 synaptic complexes were assembled in vitro in the absence of Mg(2+) and co-injected with a target plasmid into developing yellow fever mosquito, Aedes aegypti, embryos. Target plasmids recovered from embryos a day later were screened for the presence of Tn5. Recombinants (transposition events) were found at a frequency of 1.2 x 10(-3). Some transposition events did not appear to be associated with canonical 9 bp direct duplications at the site of insertion and also were associated with either deletions or rearrangements. A Tn5 element containing the brain-specific transgene, 3 x P3DsRed, was assembled into synaptic complexes in vitro and injected into pre-blastoderm embryos of Ae. aegypti. Of the approximately 900 embryos surviving injection and developing into adults, two produced transgenic progeny. Both transgenic events involved the co-integrations of approximately five elements resulting in nested and tandem arrayed Tn5::3 x P3DsRed elements. This study extends the known host range of Tn5 to insects and makes available to insect biologists and others another eukaryotic genome-manipulation tool. The hyperactivity of synaptic complexes may be responsible for the unusual clustering of elements and managing this aspect of the element's behavior will be important in future applications of this technology to insects.     

Microinjection of A. aegypti embryos to obtain transgenic mosquitoes

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue fever. The techniques for correctly preparing microinjection needles, desiccating embryos, and performing microinjection are demonstrated.     

Fossil rhabdoviral sequences integrated into arthropod genomes: ontogeny, evolution, and potential functionality

Retroelements represent a considerable fraction of many eukaryotic genomes and are considered major drives for adaptive genetic innovations. Recent discoveries showed that despite not normally using DNA intermediates like retroviruses do, Mononegaviruses (i.e., viruses with nonsegmented, negative-sense RNA genomes) can integrate gene fragments into the genomes of their hosts. This was shown for Bornaviridae and Filoviridae, the sequences of which have been found integrated into the germ line cells of many vertebrate hosts. Here, we show that Rhabdoviridae sequences, the major Mononegavirales family, have integrated only into the genomes of arthropod species. We identified 185 integrated rhabdoviral elements (IREs) coding for nucleoproteins, glycoproteins, or RNA-dependent RNA polymerases; they were mostly found in the genomes of the mosquito Aedes aegypti and the blacklegged tick Ixodes scapularis. Phylogenetic analyses showed that most IREs in A. aegypti derived from multiple independent integration events. Since RNA viruses are submitted to much higher substitution rates as compared with their hosts, IREs thus represent fossil traces of the diversity of extinct Rhabdoviruses. Furthermore, analyses of orthologous IREs in A. aegypti field mosquitoes sampled worldwide identified an integrated polymerase IRE fragment that appeared under purifying selection within several million years, which supports a functional role in the host's biology. These results show that A. aegypti was subjected to repeated Rhabdovirus infectious episodes during its evolution history, which led to the accumulation of many integrated sequences. They also suggest that like retroviruses, integrated rhabdoviral sequences may participate actively in the evolution of their hosts.     

Alternative splicing generates multiple transcripts of the inhibitor of apoptosis protein 1 in Aedes and Culex spp. mosquitoes

We determined the sequences of cDNA encoding Inhibitor of Apoptosis Protein 1 (IAP1) homologues from Aedes triseriatus, Aedes albopictus, Aedes aegypti, Culex pipiens and Culex tarsalis. The cDNAs encode translation products that share > or = 84% sequence similarity. The IAP1 mRNA of each mosquito species exists as 3-5 distinct variants due to the presence of heterogeneous sequences at the distal end of their 5'UTRs. Partial genomic sequencing upstream of the 5' end of the Ae. triseriatus IAP1 gene, and analysis of the Ae. aegypti genomic sequence, suggest that these mRNA variants are generated by alternative splicing. Each IAP1 mRNA variant from Ae. triseriatus and Cx. pipiens was detected by RT-PCR in all mosquito life-stages and adult tissues examined, and the relative concentration of each Ae. triseriatus IAP mRNA variant in various tissues was determined.     

水稻双元细菌人工染色体载体系统转化体系的建立

普通双元载体己被广泛碰用于农杆菌介导的植物转化,但这类载体通常只能转移5～20kb的外源DNA片段;而双元细菌人工染色体（BIBAC）载休可以弥补普通双元裁体的不足,通过它已在烟草、番茄等双子叶植物中实现了大片段DNA（150kb）的转移。BIBAC载体在单子叶植物转化中的应用尚未见报道。面于单、双子叶植物间以及大、小片段转化间的转化体系存在明显差异,常规的农杆菌介导的水稻转化体系不能适应BIBAC系统转化的要求。因此,建立适于BIBAC系统的水稻转化体系是十分必要的。通过比较不同的受体材料,不同的预培养、其培养条件,不同的去除农杆菌及选择阳性愈伤的方式等对转化效率的影响,建矿了适合水稻BIBAC系统的转化体系。该体系的技术要点包括：以水稻品种H1493为转化受体：以含毒性辅助质粒pCH32的LBA4404菌株（HP4404）为侵染菌株;预培养的培养拱pH5．6：以N6A代替AAM悬浮农杆菌：侵染菌液浓度为OD600=1．0;共培养温度为24℃;采用过渡（Resting）培养除去农杆菌;采用二步法进行选择等。基于PCR检测、Southern印迹分析的结果表明,BIBAC载体所携带的插入片段及标记基因已整合到转化植株的基因组中。这个体系的建立为在水稻中利用BIBAC系统进行大片段DNA转化奠定基础。     

An in vivo transfection approach elucidates a role for Aedes aegypti thioester-containing proteins in flaviviral infection

Mosquitoes transmit pathogens that cause infectious diseases of global importance. Techniques to easily introduce genes into mosquitoes, however, limit investigations of the interaction between microbes and their arthropod vectors. We now show that a cationic liposome significantly enhances delivery and expression of plasmid DNA in Aedes aegypti and Anopheles gambiae mosquitoes. We then introduced the genes for Ae. aegypti thioester-containing proteins (AeTEPs), which are involved in the control of flaviviral infection, into mosquitoes using this technique. In vivo transfection of AeTEP-1 into Ae. aegypti significantly reduced dengue virus infection, suggesting that the approach can further our understanding of pathogen-mosquito interactions.     

The dynamics of interactions between Plasmodium and the mosquito: a study of the infectivity of Plasmodium berghei and Plasmodium gallinaceum,and their transmission by Anopheles stephensi,Anopheles gambiae and Aedes aegypti

Knowledge of parasite-mosquito interactions is essential to develop strategies that will reduce malaria transmission through the mosquito vector. In this study we investigated the development of two model malaria parasites, Plasmodium berghei and Plasmodium gallinaceum, in three mosquito species Anopheles stephensi, Anopheles gambiae and Aedes aegypti. New methods to study gamete production in vivo in combination with GFP-expressing ookinetes were employed to measure the large losses incurred by the parasites during infection of mosquitoes. All three mosquito species transmitted P. gallinaceum; P. berghei was only transmitted by Anopheles spp. Plasmodium gallinaceum initiates gamete production with high efficiency equally in the three mosquito species. By contrast P. berghei is less efficiently activated to produce gametes, and in Ae. aegypti microgamete formation is almost totally suppressed. In all parasite/vector combinations ookinete development is inefficient, 500-100,000-fold losses were encountered. Losses during ookinete-to-oocyst transformation range from fivefold in compatible vector parasite combinations (P. berghei/An. stephensi), through >100-fold in poor vector/parasite combinations (P. gallinaceum/An. stephensi), to complete blockade (>1,500 fold) in others (P. berghei/Ae. aegypti). Plasmodium berghei ookinetes survive poorly in the bloodmeal of Ae. aegypti and are unable to invade the midgut epithelium. Cultured mature ookinetes of P. berghei injected directly into the mosquito haemocoele produced salivary gland sporozoites in An. stephensi, but not in Ae. aegypti, suggesting that further species-specific incompatibilities occur downstream of the midgut epithelium in Ae. aegypti. These results show that in these parasite-mosquito combinations the susceptibility to malarial infection is regulated at multiple steps during the development of the parasites. Understanding these at the molecular level may contribute to the development of rational strategies to reduce the vector competence of malarial vectors.     

Purified mariner (Mos1) transposase catalyzes the integration of marked elements into the germ-line of the yellow fever mosquito, Aedes aegypti

Derivatives of the mariner transposable element, Mos1, from Drosophila mauritiana, can integrate into the germ-line of the yellow fever mosquito, Aedes aegypti. Previously, the transposase required to mobilize Mos1 was provided in trans by a helper plasmid expressing the enzyme under the control of the D. psuedoobscura heat-shock protein 82 promoter. Here we tested whether purified recombinant Mos1 transposase could increase the recovery of Ae. aegypti transformants. Mos1 transposase was injected into white-eyed, kh(w)/kh(w), Ae. aegypti embryos with a Mos1 donor plasmid containing a copy of the wild-type allele of the D. melanogaster cinnabar gene. Transformed mosquitoes were recognized by partial restoration of eye color in the G(1) animals and confirmed by Southern analyses of genomic DNA. At Mos1 transposase concentrations approaching 100 nM, the rate of germ-line transformants arising from independent insertions in G(0) animals was elevated 2-fold compared to that seen in experiments with helper plasmids. Furthermore, the recovery of total G(1) transformants was increased 7.5-fold over the frequency seen with co-injected helper plasmid. Southern blot analyses and gene amplification experiments confirmed the integration of the transposons into the mosquito genome, although not all integrations were of the expected cut-and-paste type transposition. The increased frequency of germ-line integrations obtained with purified transposase will facilitate the generation of Mos1 transgenic mosquitoes and the application of transgenic approaches to the biology of this important vector of multiple pathogens.     

siRNA-mediated gene targeting in Aedes aegypti embryos reveals that frazzled regulates vector mosquito CNS development

Although mosquito genome projects uncovered orthologues of many known developmental regulatory genes, extremely little is known about the development of vector mosquitoes. Here, we investigate the role of the Netrin receptor frazzled (fra) during embryonic nerve cord development of two vector mosquito species. Fra expression is detected in neurons just prior to and during axonogenesis in the embryonic ventral nerve cord of Aedes aegypti (dengue vector) and Anopheles gambiae (malaria vector). Analysis of fra function was investigated through siRNA-mediated knockdown in Ae. aegypti embryos. Confirmation of fra knockdown, which was maintained throughout embryogenesis, indicated that microinjection of siRNA is an effective method for studying gene function in Ae. aegypti embryos. Loss of fra during Ae. aegypti development results in thin and missing commissural axons. These defects are qualitatively similar to those observed in Dr. melanogaster fra null mutants. However, the Aa. aegypti knockdown phenotype is stronger and bears resemblance to the Drosophila commissureless mutant phenotype. The results of this investigation, the first targeted knockdown of a gene during vector mosquito embryogenesis, suggest that although Fra plays a critical role during development of the Ae. aegypti ventral nerve cord, mechanisms regulating embryonic commissural axon guidance have evolved in distantly related insects.     

Transient and stable transformation of wheat with DNA preparations delivered by a biolistic method

An optimized procedure for transformation of wheat with the use of a Biolistic Particle Delivery System PDS 1000/He to deliver foreign DNA is described in detail. The bacterial uidA and bar genes (both driven by plant promoters) were utilized as the reporter and selectable marker genes, respectively. Moderately high gas pressure appeared to be most important to achieve the highest level of transient GUS expression in target tissues. There was, however, no apparent correlation between transient and stable GUS expression. The presence of telomeric DNA sequences in an uidA gene-containing vector did not influence transient GUS expression but, apparently, prevented its stable expression. Mechanical lesions caused by the bombardment (tungsten particles) seemed to be less severe when embryo- derived calli, instead of freshly excised immature embryos, were used as the target tissue. The limited ability of callus cells for regeneration, together with a restricted number of cells that receive the foreign DNA by particle bombardment, result in a low efficiency of wheat stable transformation.