50 results found
Bernardini F, Kriezis A, Galizi R, et al., 2019, Introgression of a synthetic sex ratio distortion system from Anopheles gambiae into Anopheles arabiensis (vol 9, 5158, 2019), SCIENTIFIC REPORTS, Vol: 9, ISSN: 2045-2322
Bernardini F, Kriezis A, Galizi R, et al., 2019, Introgression of a synthetic sex ratio distortion system from Anopheles gambiae into Anopheles arabiensis, Scientific Reports, Vol: 9, ISSN: 2045-2322
I-PpoI is a homing endonuclease that has a high cleavage activity and specificity for a conserved sequence within the ribosomal rDNA repeats, located in a single cluster on the Anopheles gambiae X chromosome. This property has been exploited to develop a synthetic sex ratio distortion system in this mosquito species. When I-PpoI is expressed from a transgene during spermatogenesis in mosquitoes, the paternal X chromosome is shredded and only Y chromosome-bearing sperm are viable, resulting in a male-biased sex ratio of >95% in the progeny. These distorter male mosquitoes can efficiently suppress caged wild-type populations, providing a powerful tool for vector control strategies. Given that malaria mosquito vectors belong to a species complex comprising at least two major vectors, we investigated whether the sex distorter I-PpoI, originally integrated in the A. gambiae genome, could be transferred via introgression to the sibling vector species Anopheles arabiensis. In compliance with Haldane’s rule, F1 hybrid male sterility is known to occur in all intercrosses among members of the Anopheles gambiae complex. A scheme based on genetic crosses and transgene selection was used to bypass F1 hybrid male sterility and introgress the sex distorter I-PpoI into the A. arabiensis genetic background. Our data suggest that this sex distortion technique can be successfully applied to target A. arabiensis mosquitoes.
Bernardini F, Haghighat-Khah RE, Galizi R, et al., 2018, Molecular tools and genetic markers for the generation of transgenic sexing strains in Anopheline mosquitoes, Parasites & Vectors, Vol: 11, ISSN: 1756-3305
Malaria is a serious global health burden, affecting more than 200 million people each year in over 90 countries, predominantly in Africa, Asia and the Americas. Since the year 2000, a concerted effort to combat malaria has reduced its incidence by more than 40%, primarily due to the use of insecticide-treated bednets, indoor residual spraying and artemisinin-based combination drug therapies. Nevertheless, the cost of control is expected to nearly triple over the next decade and the current downward trend in disease transmission is threatened by the rise of resistance to drugs and insecticides. Novel strategies that are sustainable and cost-effective are needed to help usher in an era of malaria elimination. The most effective strategies thus far have focussed on control of the mosquito vector. The sterile insect technique (SIT) is a potentially powerful strategy that aims to suppress mosquito populations through the unproductive mating of wild female mosquitoes with sterile males that are released en masse. The technique and its derivatives are currently not appropriate for malaria control because it is difficult to sterilise males without compromising their ability to mate, and because anopheline males cannot be easily separated from females, which if released, could contribute to disease transmission. Advances in genome sequencing technologies and the development of transgenic techniques provide the tools necessary to produce mosquito sexing strains, which promise to improve current malaria-control programs and pave the way for new ones. In this review, the progress made in the development of transgenic sexing strains for the control of Anopheles gambiae, a major vector of human malaria, is discussed.
Kyrou K, Hammond AM, Galizi R, et al., 2018, A CRISPR-Cas9 gene drive targeting doublesex causes complete population suppression in caged Anopheles gambiae mosquitoes, Nature Biotechnology, Vol: 36, Pages: 1062-1066, ISSN: 1087-0156
In the human malaria vector Anopheles gambiae, the gene doublesex (Agdsx) encodes two alternatively spliced transcripts, dsx-female (AgdsxF) and dsx-male (AgdsxM), that control differentiation of the two sexes. The female transcript, unlike the male, contains an exon (exon 5) whose sequence is highly conserved in all Anopheles mosquitoes so far analyzed. We found that CRISPR–Cas9-targeted disruption of the intron 4–exon 5 boundary aimed at blocking the formation of functional AgdsxF did not affect male development or fertility, whereas females homozygous for the disrupted allele showed an intersex phenotype and complete sterility. A CRISPR–Cas9 gene drive construct targeting this same sequence spread rapidly in caged mosquitoes, reaching 100% prevalence within 7–11 generations while progressively reducing egg production to the point of total population collapse. Owing to functional constraint of the target sequence, no selection of alleles resistant to the gene drive occurred in these laboratory experiments. Cas9-resistant variants arose in each generation at the target site but did not block the spread of the drive.
Hammond A, Kyrou K, Gribble M, et al., 2018, Improved CRISPR-based suppression gene drives mitigate resistance and impose a large reproductive load on laboratory-contained mosquito populations, Publisher: Cold Spring Harbor Laboratory
<jats:title>Abstract</jats:title><jats:p>CRISPR-based genes drives bias their own inheritance and can be used to modify entire populations of insect vectors of disease as a novel form of sustainable disease control. Gene drives designed to interfere with female fertility can suppress populations of the mosquito vector of malaria, however laboratory demonstrations showed strong unintended fitness costs and high levels of resistant mutations that limited the potential of the first generation of gene drives to spread. We describe three new gene drives designed to restrict spatio-temporal nuclease expression by using novel regulatory sequences. Two of the three new designs dramatically improve fitness and mitigate the creation and selection of resistance. We dissect the relative contributions of germline CRISPR activity versus embryonic CRISPR activity resulting from parental deposition, showing that the improved performance of the new designs is due to tighter germline restriction of the nuclease activity and significantly lower rates of end-joining repair in the embryo. Moreover, we demonstrate in laboratory-contained population experiments that these gene drives show remarkably improved invasion dynamics compared to the first generation drives, resulting in greater than 90% suppression of the reproductive output and a delay in the emergence of target site resistance, even at a loosely constrained target sequence. These results illustrate important considerations for gene drive design and will help expedite the development of gene drives designed to control malaria transmission in Africa.</jats:p>
Quinlan MM, Birungi J, Coulibaly MB, et al., 2018, Containment studies of transgenic mosquitoes in disease endemic countries: the broad concept of facilities readiness, Vector-Borne and Zoonotic Diseases, Vol: 18, Pages: 14-20, ISSN: 1530-3667
Genetic strategies for large scale pest or vector control using modified insects are not yet operational in Africa, and currently rely on import of the modified strains to begin preliminary, contained studies. Early involvement of research teams from participating countries is crucial to evaluate candidate field interventions. Following the recommended phased approach for novel strategies, evaluation should begin with studies in containment facilities. Experiences to prepare facilities and build international teams for research on transgenic mosquitoes revealed some important organizing themes underlying the concept of “facilities readiness,” or the point at which studies in containment may proceed, in sub-Saharan African settings. First, “compliance” for research with novel or non-native living organisms was defined as the fulfillment of all legislative and regulatory requirements. This is not limited to regulations regarding use of transgenic organisms. Second, the concept of “colony utility” was related to the characteristics of laboratory colonies being produced so that results of studies may be validated across time, sites, and strains or technologies; so that the appropriate candidate strains are moved forward toward field studies. Third, the importance of achieving “defensible science” was recognized, including that study conclusions can be traced back to evidence, covering the concerns of various stakeholders over the long term. This, combined with good stewardship of resources and appropriate funding, covers a diverse set of criteria for declaring when “facilities readiness” has been attained. It is proposed that, despite the additional demands on time and resources, only with the balance of and rigorous achievement of each of these organizing themes can collaborative research into novel strategies in vector or pest control reliably progress past initial containment studies.
Nolan T, 2017, How to handle gene drives in arthropods?, Pathog Glob Health, Vol: 111, Pages: 403-403
Lombardo F, Salvemini M, Fiorillo C, et al., 2017, Deciphering the olfactory repertoire of the tiger mosquito Aedes albopictus, BMC Genomics, Vol: 18, ISSN: 1471-2164
BackgroundThe Asian tiger mosquito Aedes albopictus is a highly invasive species and competent vector of several arboviruses (e.g. dengue, chikungunya, Zika) and parasites (e.g. dirofilaria) of public health importance. Compared to other mosquito species, Ae. albopictus females exhibit a generalist host seeking as well as a very aggressive biting behaviour that are responsible for its high degree of nuisance. Several complex mosquito behaviours such as host seeking, feeding, mating or oviposition rely on olfactory stimuli that target a range of sensory neurons localized mainly on specialized head appendages such as antennae, maxillary palps and the mouthparts.ResultsWith the aim to describe the Ae. albopictus olfactory repertoire we have used RNA-seq to reveal the transcriptome profiles of female antennae and maxillary palps. Male heads and whole female bodies were employed as reference for differential expression analysis. The relative transcript abundance within each tissue (TPM, transcripts per kilobase per million) and the pairwise differential abundance in the different tissues (fold change values and false discovery rates) were evaluated. Contigs upregulated in the antennae (620) and maxillary palps (268) were identified and relative GO and PFAM enrichment profiles analysed. Chemosensory genes were described: overall, 77 odorant binding proteins (OBP), 82 odorant receptors (OR), 60 ionotropic receptors (IR) and 30 gustatory receptors (GR) were identified by comparative genomics and transcriptomics. In addition, orthologs of genes expressed in the female/male maxillary palps and/or antennae and involved in thermosensation (e.g. pyrexia and arrestin1), mechanosensation (e.g. piezo and painless) and neuromodulation were classified.ConclusionsWe provide here the first detailed transcriptome of the main Ae. albopictus sensory appendages, i.e. antennae and maxillary palps. A deeper knowledge of the olfactory repertoire of the tiger mosquito will help to better under
Hammond AM, Kyrou K, Bruttini M, et al., 2017, The creation and selection of mutations resistant to a gene drive over multiple generations in the malaria mosquito., PLoS Genetics, Vol: 13, ISSN: 1553-7390
Gene drives have enormous potential for the control of insect populations of medical and agricultural relevance. By preferentially biasing their own inheritance, gene drives can rapidly introduce genetic traits even if these confer a negative fitness effect on the population. We have recently developed gene drives based on CRISPR nuclease constructs that are designed to disrupt key genes essential for female fertility in the malaria mosquito. The construct copies itself and the associated genetic disruption from one homologous chromosome to another during gamete formation, a process called homing that ensures the majority of offspring inherit the drive. Such drives have the potential to cause long-lasting, sustainable population suppression, though they are also expected to impose a large selection pressure for resistance in the mosquito. One of these population suppression gene drives showed rapid invasion of a caged population over 4 generations, establishing proof of principle for this technology. In order to assess the potential for the emergence of resistance to the gene drive in this population we allowed it to run for 25 generations and monitored the frequency of the gene drive over time. Following the initial increase of the gene drive we observed a gradual decrease in its frequency that was accompanied by the spread of small, nuclease-induced mutations at the target gene that are resistant to further cleavage and restore its functionality. Such mutations showed rates of increase consistent with positive selection in the face of the gene drive. Our findings represent the first documented example of selection for resistance to a synthetic gene drive and lead to important design recommendations and considerations in order to mitigate for resistance in future gene drive applications.
Bernardini F, Galizi R, Wunderlich M, et al., 2017, Cross-Species Y Chromosome Function Between Malaria Vectors of the Anopheles gambiae Species Complex., Genetics, ISSN: 0016-6731
Y chromosome function, structure and evolution is poorly understood in many species including the Anopheles genus of mosquitoes, an emerging model system for studying speciation that also represents the major vectors of malaria. While the Anopheline Y had previously been implicated in male mating behavior, recent data from the Anopheles gambiae complex suggests that, apart from the putative primary sex-determiner, no other genes are conserved on the Y. Studying the functional basis of the evolutionary divergence of the Y chromosome in the gambiae complex is complicated by complete F1 male hybrid sterility. Here we used an F1xF0 crossing scheme to overcome a severe bottleneck of male hybrid incompatibilities and enabled us to experimentally purify a genetically labelled A. gambiae Y chromosome in an A. arabiensis background. Whole genome sequencing confirmed that the A. gambiae Y retained its original sequence content in the A. arabiensis genomic background. In contrast to comparable experiments in Drosophila, we find that the presence of a heterospecific Y chromosome has no significant effect on the expression of A. arabiensis genes and transcriptional differences can be explained almost exclusively as a direct consequence of transcripts arising from sequence elements present on the A. gambiae Y chromosome itself. We find that Y hybrids show no obvious fertility defects and no substantial reduction in male competitiveness. Our results demonstrate that, despite their radically different structure, Y chromosomes of these two species of the gambiae complex that diverged an estimated 1.85Myr ago function interchangeably, thus indicating that the Y chromosome does not harbor loci contributing to hybrid incompatibility. Therefore, Y chromosome gene flow between members of the gambiae complex is possible even at their current level of divergence. Importantly, this also suggests that malaria control interventions based on sex-distorting Y drive would be transferable, whethe
Werther R, Hallinan JP, Lambert AR, et al., 2017, Crystallographic analyses illustrate significant plasticity and efficient recoding of meganuclease target specificity, Nucleic Acids Research, Vol: 45, Pages: 8621-8634, ISSN: 0305-1048
The retargeting of protein–DNA specificity, outsideof extremely modular DNA binding proteins suchas TAL effectors, has generally proved to be quitechallenging. Here, we describe structural analysesof five different extensively retargeted variants of asingle homing endonuclease, that have been shownto function efficiently in ex vivo and in vivo applications.The redesigned proteins harbor mutationsat up to 53 residues (18%) of their amino acid sequence,primarily distributed across the DNA bindingsurface, making them among the most signifi-cantly reengineered ligand-binding proteins to date.Specificity is derived from the combined contributionsof DNA-contacting residues and of neighboringresidues that influence local structural organization.Changes in specificity are facilitated by theability of all those residues to readily exchange bothform and function. The fidelity of recognition is notprecisely correlated with the fraction or total numberof residues in the protein–DNA interface that areactually involved in DNA contacts, including directionalhydrogen bonds. The plasticity of the DNArecognitionsurface of this protein, which allows substantialretargeting of recognition specificity withoutrequiring significant alteration of the surroundingprotein architecture, reflects the ability of the correspondinggenetic elements to maintain mobility andpersistence in the face of genetic drift within potentialhost target sites.
Papa F, Windbichler N, Waterhouse RM, et al., 2017, Rapid evolution of female-biased genes among four species of Anopheles malaria mosquitoes, GENOME RESEARCH, Vol: 27, Pages: 1536-1548, ISSN: 1088-9051
Understanding how phenotypic differences between males and females arise from the sex-biased expression of nearly identical genomes can reveal important insights into the biology and evolution of a species. Among Anopheles mosquito species, these phenotypic differences include vectorial capacity, as it is only females that blood feed and thus transmit human malaria. Here, we use RNA-seq data from multiple tissues of four vector species spanning the Anopheles phylogeny to explore the genomic and evolutionary properties of sex-biased genes. We find that, in these mosquitoes, in contrast to what has been found in many other organisms, female-biased genes are more rapidly evolving in sequence, expression, and genic turnover than male-biased genes. Our results suggest that this atypical pattern may be due to the combination of sex-specific life history challenges encountered by females, such as blood feeding. Furthermore, female propensity to mate only once in nature in male swarms likely diminishes sexual selection of post-reproductive traits related to sperm competition among males. We also develop a comparative framework to systematically explore tissue- and sex-specific splicing to document its conservation throughout the genus and identify a set of candidate genes for future functional analyses of sex-specific isoform usage. Finally, our data reveal that the deficit of male-biased genes on the X Chromosomes in Anopheles is a conserved feature in this genus and can be directly attributed to chromosome-wide transcriptional regulation that de-masculinizes the X in male reproductive tissues.
Bernardini F, Galizi R, Wunderlich M, et al., 2017, Cross-species Y chromosome function between malaria vectors of the Anopheles gambiae species complex
<jats:title>Abstract</jats:title><jats:p>Y chromosome function, structure and evolution is poorly understood in many species including the <jats:italic>Anopheles</jats:italic> genus of mosquitoes, an emerging model system for studying speciation that also represents the major vectors of malaria. While the Anopheline Y had previously been implicated in male mating behavior, recent data from the <jats:italic>Anopheles gambiae</jats:italic> complex suggests that, apart from the putative primary sex-determiner, no other genes are conserved on the Y. Studying the functional basis of the evolutionary divergence of the Y chromosome in the gambiae complex is complicated by complete F1 male hybrid sterility. Here we used an F1xF0 crossing scheme to overcome a severe bottleneck of male hybrid incompatibilities and enabled us to experimentally purify a genetically labelled <jats:italic>A. gambiae</jats:italic> Y chromosome in an <jats:italic>A. arabiensis</jats:italic> background. Whole genome sequencing confirmed that the <jats:italic>A. gambiae</jats:italic> Y retained its original sequence content in the <jats:italic>A. arabiensis</jats:italic> genomic background. In contrast to comparable experiments in <jats:italic>Drosophila</jats:italic>, we find that the presence of a heterospecific Y chromosome has no significant effect on the expression of <jats:italic>A. arabiensis</jats:italic> genes and transcriptional differences can be explained almost exclusively as a direct consequence of transcripts arising from sequence elements present on the <jats:italic>A. gambiae</jats:italic> Y chromosome itself. We find that Y hybrids show no obvious fertility defects and no substantial reduction in male competitiveness. Our results demonstrate that, despite their radically different structure, Y chromosomes of these two species of the gambiae complex that diver
Hammond A, Kyrou K, Bruttini M, et al., 2017, The creation and selection of mutations resistant to a gene drive over multiple generations in the malaria mosquito
<jats:title>ABSTRACT</jats:title><jats:p>Gene drives have enormous potential for the control of insect populations of medical and agricultural relevance. By preferentially biasing their own inheritance gene drives can rapidly introduce genetic traits even if these confer a negative fitness on the population.</jats:p><jats:p>We have recently developed gene drives based on a CRISPR nuclease construct that is designed to disrupt key genes essential for female fertility in the malaria mosquito. The construct copies itself and the associated genetic disruption from one homologous chromosome to another during gamete formation, in a process called homing that ensures the majority of offspring inherit the drive. Such drives have the potential to cause long-lasting, sustainable population suppression though they are also expected to impose a large selection pressure for resistance in the mosquito. One of these population suppression gene drives showed rapid invasion of a caged population over 4 generations, establishing proof of principle for this technology. In order to assess the potential for the emergence of resistance to the gene drive in this population we allowed it to run for 25 generations and monitored the frequency of the gene drive over time. Following the initial increase of the gene drive we noticed a gradual decrease in its frequency that was accompanied emergence of small, nuclease-induced mutations at the target gene that are resistant to further cleavage and restore its functionality. Such mutations show rates of increase consistent with positive selection in the face of the gene drive. Our findings represent the first documented example of selection for resistance to a synthetic gene drive and lead to important design recommendations and considerations in order to mitigate for resistance for future gene drive applications.</jats:p>
NOLAN T, CRISANTI A, 2017, Using gene drives to limit the spread of malaria, Scientist, Vol: 31, ISSN: 0890-3670
Beaghton A, Hammond A, Nolan T, et al., 2017, Requirements for Driving Antipathogen Effector Genes into Populations of Disease Vectors by Homing, Genetics, Vol: 205, Pages: 1587-1596
Simoes ML, Dong Y, Hammond A, et al., 2017, The Anopheles FBN9 immune factor mediates Plasmodium species-specific defense through transgenic fat body expression, Developmental and Comparative Immunology, Vol: 67, Pages: 257-265
Galizi R, Hammond A, Kyrou K, et al., 2016, A CRISPR-Cas9 sex-ratio distortion system for genetic control., Scientific Reports, Vol: 6, ISSN: 2045-2322
Genetic control aims to reduce the ability of insect pest populations to cause harm via the release of modified insects. One strategy is to bias the reproductive sex ratio towards males so that a population decreases in size or is eliminated altogether due to a lack of females. We have shown previously that sex ratio distortion can be generated synthetically in the main human malaria vector Anopheles gambiae, by selectively destroying the X-chromosome during spermatogenesis, through the activity of a naturally-occurring endonuclease that targets a repetitive rDNA sequence highly-conserved in a wide range of organisms. Here we describe a CRISPR-Cas9 sex distortion system that targets ribosomal sequences restricted to the member species of the Anopheles gambiae complex. Expression of Cas9 during spermatogenesis resulted in RNA-guided shredding of the X-chromosome during male meiosis and produced extreme male bias among progeny in the absence of any significant reduction in fertility. The flexibility of CRISPR-Cas9 combined with the availability of genomic data for a range of insects renders this strategy broadly applicable for the species-specific control of any pest or vector species with an XY sex-determination system by targeting sequences exclusive to the female sex chromosome.
Hall AB, Papathanos P-A, Sharma A, et al., 2016, Radical remodeling of the Y chromosome in a recent radiation of malaria mosquitoes, Proceedings of the National Academy of Sciences of the United States of America, Vol: 113, Pages: E2114-E2123
Nolan T, Crisanti A, 2016, DRIVING OUT MALARIA, Scientist, Vol: 31, Pages: 24-31
Hammond A, Galizi R, Kyrou K, et al., 2015, A CRISPR-Cas9 gene drive system-targeting female reproduction in the malaria mosquito vector Anopheles gambiae, Nature Biotechnology, Vol: 34, Pages: 78-83, ISSN: 1087-0156
Gene drive systems that enable super-Mendelian inheritance of a transgene have the potential to modify insect populations over a timeframe of a few years. We describe CRISPR-Cas9 endonuclease constructs that function as gene drive systems in Anopheles gambiae, the main vector for malaria. We identified three genes (AGAP005958, AGAP011377 and AGAP007280) that confer a recessive female-sterility phenotype upon disruption, and inserted into each locus CRISPR-Cas9 gene drive constructs designed to target and edit each gene. For each targeted locus we observed a strong gene drive at the molecular level, with transmission rates to progeny of 91.4 to 99.6%. Population modeling and cage experiments indicate that a CRISPR-Cas9 construct targeting one of these loci, AGAP007280, meets the minimum requirement for a gene drive targeting female reproduction in an insect population. These findings could expedite the development of gene drives to suppress mosquito populations to levels that do not support malaria transmission.
Volohonsky G, Terenzi O, Soichot J, et al., 2015, Tools for Anopheles gambiae Transgenesis, G3-Genes Genomes Genetics, Vol: 5, Pages: 1151-1163
Castellano L, Rizzi E, Krell J, et al., 2015, The germline of the malaria mosquito produces abundant miRNAs, endo-siRNAs, piRNAs and 29-nt small RNAs, Bmc Genomics, Vol: 16
Neafsey DE, Waterhouse RM, Abai MR, et al., 2015, Highly evolvable malaria vectors: The genomes of 16 Anopheles mosquitoes, Science, Vol: 347
Thyme SB, Boissel SJS, Quadri SA, et al., 2014, Reprogramming homing endonuclease specificity through computational design and directed evolution, Nucleic Acids Research, Vol: 42, Pages: 2564-2576, ISSN: 0305-1048
Homing endonucleases (HEs) can be used to induce targeted genome modification to reduce the fitness of pathogen vectors such as the malaria-transmitting Anopheles gambiae and to correct deleterious mutations in genetic diseases. We describe the creation of an extensive set of HE variants with novel DNA cleavage specificities using an integrated experimental and computational approach. Using computational modeling and an improved selection strategy, which optimizes specificity in addition to activity, we engineered an endonuclease to cleave in a gene associated with Anopheles sterility and another to cleave near a mutation that causes pyruvate kinase deficiency. In the course of this work we observed unanticipated context-dependence between bases which will need to be mechanistically understood for reprogramming of specificity to succeed more generally.
Fuchs S, Rende E, Crisanti A, et al., 2014, Disruption of aminergic signalling reveals novel compounds with distinct inhibitory effects on mosquito reproduction, locomotor function and survival, Scientific Reports, Vol: 4
Fuchs S, Rende E, Crisanti A, et al., 2014, Disruption of aminergic signalling reveals novel compounds with distinct inhibitory effects on mosquito reproduction, locomotor function and survival., Scientific Reports, Vol: 4, Pages: srep05526-srep05526
Hammond AM, Nolan T, Benedict MQ, 2014, Sex-, tissue- and stage-specific transgene expression., Transgenic insects: techniques and applications, Pages: 29-50
Fuchs S, Behrends V, Bundy JG, et al., 2014, Phenylalanine Metabolism Regulates Reproduction and Parasite Melanization in the Malaria Mosquito, Plos One, Vol: 9
Fuchs S, Nolan T, Crisanti A, 2013, Mosquito transgenic technologies to reduce plasmodium transmission, Methods in Molecular Biology, Vol: 923, Pages: 601-622, ISSN: 1064-3745
The ability to introduce genetic constructs of choice into the genome of Anopheles mosquitoes provides a valuable tool to study the molecular interactions between the Plasmodium parasite and its insect host. In the long term, this technology could potentially offer new ways to control vector-borne diseases through the suppression of target mosquito populations or through the introgression of traits that preclude pathogen transmission. Here, we describe in detail protocols for the generation of transgenic Anopheles gambiae mosquitoes based on germ-line transformation using either modified transposable elements or the sitespecific PhiC31 recombinase .© Springer Science+Business Media, LLC 2013.
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