Site-Specific Integration and Expression of an Anti-Malarial Gene in Transgenic Anopheles gambiae Significantly Reduces Plasmodium Infections
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{"title"=>"Site-specific integration and expression of an anti-malarial gene in transgenic Anopheles gambiae significantly reduces Plasmodium infections", "type"=>"journal", "authors"=>[{"first_name"=>"Janet M.", "last_name"=>"Meredith", "scopus_author_id"=>"7102609450"}, {"first_name"=>"Sanjay", "last_name"=>"Basu", "scopus_author_id"=>"37661111000"}, {"first_name"=>"Derric D.", "last_name"=>"Nimmo", "scopus_author_id"=>"12798223100"}, {"first_name"=>"Isabelle", "last_name"=>"Larget-Thiery", "scopus_author_id"=>"6507576906"}, {"first_name"=>"Emma L.", "last_name"=>"Warr", "scopus_author_id"=>"8684370800"}, {"first_name"=>"Ann", "last_name"=>"Underhill", "scopus_author_id"=>"17342814600"}, {"first_name"=>"Clare C.", "last_name"=>"McArthur", "scopus_author_id"=>"37661905900"}, {"first_name"=>"Victoria", "last_name"=>"Carter", "scopus_author_id"=>"57197152718"}, {"first_name"=>"Hilary", "last_name"=>"Hurd", "scopus_author_id"=>"7006852961"}, {"first_name"=>"Catherine", "last_name"=>"Bourgouin", "scopus_author_id"=>"6603717350"}, {"first_name"=>"Paul", "last_name"=>"Eggleston", "scopus_author_id"=>"7102797750"}], "year"=>2011, "source"=>"PLoS ONE", "identifiers"=>{"scopus"=>"2-s2.0-79551536218", "sgr"=>"79551536218", "issn"=>"19326203", "doi"=>"10.1371/journal.pone.0014587", "pmid"=>"21283619", "isbn"=>"1932-6203", "pui"=>"361204237"}, "id"=>"0516b6f1-2def-3744-9ac0-47cec27e49e6", "abstract"=>"Diseases transmitted by mosquitoes have a devastating impact on global health and this is worsening due to difficulties with existing control measures and climate change. Genetically modified mosquitoes that are refractory to disease transmission are seen as having great potential in the delivery of novel control strategies. Historically the genetic modification of insects has relied upon transposable elements which have many limitations despite their successful use. To circumvent these limitations the Streptomyces phage phiC31 integrase system has been successfully adapted for site-specific transgene integration in insects. Here, we present the first site-specific transformation of Anopheles gambiae, the principal vector of human malaria. Mosquitoes were initially engineered to incorporate the phiC31 targeting site at a defined genomic location. A second phase of genetic modification then achieved site-specific integration of Vida3, a synthetic anti-malarial gene. Expression of Vida3, specifically in the midgut of bloodfed females, offered consistent and significant protection against Plasmodium yoelii nigeriensis, reducing average parasite intensity by 85%. Similar protection was observed against Plasmodium falciparum in some experiments, although protection was inconsistent. In the fight against malaria, it is imperative to establish a broad repertoire of both anti-malarial effector genes and tissue-specific promoters for their expression, enabling those offering maximum effect with minimum fitness cost to be identified. In the future, this technology will allow effective comparisons and informed choices to be made, potentially leading to complete transmission blockade.", "link"=>"http://www.mendeley.com/research/sitespecific-integration-expression-antimalarial-gene-transgenic-anopheles-gambiae-significantly-red", "reader_count"=>68, "reader_count_by_academic_status"=>{"Professor > Associate Professor"=>1, "Librarian"=>1, "Researcher"=>19, "Student > Ph. D. Student"=>22, "Student > Postgraduate"=>2, "Student > Master"=>7, "Other"=>3, "Student > Bachelor"=>8, "Lecturer"=>1, "Professor"=>3, "Unspecified"=>1}, "reader_count_by_user_role"=>{"Professor > Associate Professor"=>1, "Librarian"=>1, "Researcher"=>19, "Student > Ph. D. Student"=>22, "Student > Postgraduate"=>2, "Student > Master"=>7, "Other"=>3, "Student > Bachelor"=>8, "Lecturer"=>1, "Professor"=>3, "Unspecified"=>1}, "reader_count_by_subject_area"=>{"Unspecified"=>3, "Environmental Science"=>4, "Biochemistry, Genetics and Molecular Biology"=>12, "Agricultural and Biological Sciences"=>40, "Medicine and Dentistry"=>5, "Social Sciences"=>3, "Economics, Econometrics and Finance"=>1}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>5}, "Social Sciences"=>{"Social Sciences"=>3}, "Economics, Econometrics and Finance"=>{"Economics, Econometrics and Finance"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>40}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>12}, "Unspecified"=>{"Unspecified"=>3}, "Environmental Science"=>{"Environmental Science"=>4}}, "reader_count_by_country"=>{"Pakistan"=>1, "United States"=>3, "Senegal"=>1, "Brazil"=>1, "United Kingdom"=>2, "France"=>1}, "group_count"=>6}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/804656"], "description"=>"<p>Details of mouse infections (parasitaemia and exflagellation), numbers of mosquitoes dissected (n), median and inter-quartile range of intensity of infections for experiments 1 to 5 and the pooled data.</p>", "links"=>[], "tags"=>["infections", "transgenic", "strains"], "article_id"=>475012, "categories"=>["Biotechnology", "Genetics", "Infectious Diseases"], "users"=>["Janet M. Meredith", "Sanjay Basu", "Derric D. Nimmo", "Isabelle Larget-Thiery", "Emma L. Warr", "Ann Underhill", "Clare C. McArthur", "Victoria Carter", "Hilary Hurd", "Catherine Bourgouin", "Paul Eggleston"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0014587.t001", "stats"=>{"downloads"=>2, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_P_y_nigeriensis_infections_of_transgenic_strains_E_and_EVida3_/475012", "title"=>"<i>P. y. nigeriensis</i> infections of transgenic strains E and EVida3.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2011-01-25 01:23:32"}
  • {"files"=>["https://ndownloader.figshare.com/files/804185"], "description"=>"<p>(A) Transgene organisation of the <i>piggyBac</i> insertion from pBac[3xP3-ECFPaf]-attP (not to scale) showing an unoccupied <i>attP</i> target site, ECFP marker controlled by the <i>D. melanogaster</i> 3xP3 eye-specific promoter and <i>piggyBac</i> left and right terminal inverted repeats (<i>pB</i>-L and <i>pB</i>-R). The double headed arrow identifies the probe used in Southern blot analysis, excised using <i>Eco</i>RV and <i>Pst</i>I sites, and single arrows the location of PCR primers to amplify a 391 bp fragment spanning <i>attP</i>. (B) Fluorescence profiles of transgenic strain E, G and H larvae. For all strains: i, dorsal bright-field image; ii, dorsal ECFP image; iii, ventral bright-field image; iv, ventral ECFP image. In addition to eyes and optic nerves (all strains), fluorescence is visible in cerebral ganglia and anal papillae (strains E and H) and ventral nerve ganglia (strain H). (C) Southern blot analysis of phase 1 targeting strains E, G, H and C. Genomic DNA is either uncut (1) or digested with <i>Xmn</i>I (2), <i>Nsi</i>I (3) or <i>Pst</i>I (4) and probed with the 2195 bp <i>Eco</i>RV/<i>Pst</i>I fragment described above. The band generated following <i>Nsi</i>I digestion of strain C genomic DNA most likely represents a doublet.</p>", "links"=>[], "tags"=>["genomic", "targeting"], "article_id"=>474545, "categories"=>["Biotechnology", "Genetics", "Infectious Diseases"], "users"=>["Janet M. Meredith", "Sanjay Basu", "Derric D. Nimmo", "Isabelle Larget-Thiery", "Emma L. Warr", "Ann Underhill", "Clare C. McArthur", "Victoria Carter", "Hilary Hurd", "Catherine Bourgouin", "Paul Eggleston"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0014587.g001", "stats"=>{"downloads"=>1, "page_views"=>6, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Generation_and_genomic_analysis_of_attP_targeting_strains_/474545", "title"=>"Generation and genomic analysis of <i>attP</i> targeting strains.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-01-25 01:15:45"}
  • {"files"=>["https://ndownloader.figshare.com/files/804705"], "description"=>"<p>Details of gametocyte infections, male ratio, numbers of mosquitoes dissected (n), median and inter-quartile range of intensity of infections for experiments 1 to 8.</p>", "links"=>[], "tags"=>["infections", "strains"], "article_id"=>475064, "categories"=>["Biotechnology", "Genetics", "Infectious Diseases"], "users"=>["Janet M. Meredith", "Sanjay Basu", "Derric D. Nimmo", "Isabelle Larget-Thiery", "Emma L. Warr", "Ann Underhill", "Clare C. McArthur", "Victoria Carter", "Hilary Hurd", "Catherine Bourgouin", "Paul Eggleston"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0014587.t002", "stats"=>{"downloads"=>1, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Infection_data_for_P_falciparum_infections_of_strains_E_and_EVida3_/475064", "title"=>"Infection data for <i>P. falciparum</i> infections of strains E and EVida3.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2011-01-25 01:24:24"}
  • {"files"=>["https://ndownloader.figshare.com/files/804289"], "description"=>"<p>(A) Organisation of the occupied target site (not to scale) following site-specific integration of pBattB-DsRed2-AgCP-vida tet and resolution of <i>attB</i> and <i>attP</i> into <i>attL</i> and <i>attR</i>. The insertion is flanked by phase 1-specific left and right <i>piggyBac</i> terminal inverted repeats (<i>pB</i>-L and <i>pB</i>-R) and the ECFP fluorescent marker. Vida3 is expressed from the A<i>n. gambiae</i> carboxypeptidase (<i>AgCP</i>) promoter and marked by 3xP3:DsRed2. The transcribed region (expanded) includes the <i>AgCP</i> 5′ and 3′ UTRs (light grey narrow boxes) and signal peptide (light grey wider box) and four copies of Vida3 (dark grey boxes), separated by linker sequences (white boxes). Arrows identify PCR primers that amplify fragments specific to <i>attL</i> and <i>attR</i>. (B) Fluorescence profiles of EVida3 phase 2 larvae; i, dorsal bright-field image; ii, dorsal ECFP image; iii, dorsal DsRed2 image; iv, ventral bright-field image; v, ventral ECFP image; vi, ventral DsRed2 image. In addition to eyes and optic nerves fluorescence is visible in cerebral ganglia, anal papillae and ventral nerve ganglia. Following integration in phase 2, strain E exhibits fluorescence in ventral nerve ganglia, which was not clearly visible in phase 1. This may reflect some degree of cross-talk between the two filter sets given the high intensity of the red fluorophore. Note also that DsRed2 expression is restricted to the cell nuclei by a nuclear localisation signal. (C) Confirmation of site-specific transgene integration in EVida3. PCR reactions with no template, strain E DNA or strain EVida3 DNA were established with specific primers for <i>attL</i> (301 bp, lanes 1, 2 and 3), <i>attR</i> (224 bp, lanes 4, 5 and 6) or <i>attP</i> (391 bp, lanes 7, 8 and 9).</p>", "links"=>[], "tags"=>["organisation", "characterisation", "site-specific"], "article_id"=>474647, "categories"=>["Biotechnology", "Genetics", "Infectious Diseases"], "users"=>["Janet M. Meredith", "Sanjay Basu", "Derric D. Nimmo", "Isabelle Larget-Thiery", "Emma L. Warr", "Ann Underhill", "Clare C. McArthur", "Victoria Carter", "Hilary Hurd", "Catherine Bourgouin", "Paul Eggleston"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0014587.g002", "stats"=>{"downloads"=>1, "page_views"=>8, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Genome_organisation_and_characterisation_of_phase_2_site_specific_integrations_/474647", "title"=>"Genome organisation and characterisation of phase 2 site-specific integrations.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-01-25 01:17:27"}
  • {"files"=>["https://ndownloader.figshare.com/files/804516"], "description"=>"<p>(A) Boxplots show intensity for strains E (white boxes) and EVida3 (grey boxes) from five independent experiments and the pooled data. Oocysts and melanised ookinetes were scored as invading parasites. Vertical lines denote the 95% confidence interval, horizontal lines with symbol mark the median and the interquartile range of the data is boxed. Probabilities for significant reductions in intensity in EVida3 (*) in experiments are: 1, <i>P</i> = 0.008; 2, <i>P</i> = 0.0007; 3, <i>P</i> = 0.009; 4, <i>P</i><0.0001; 5, <i>P</i><0.0001 and pooled data 1–5, <i>P</i><0.0001. (B) Histograms show prevalence for strains E (white bars) and EVida3 (grey bars) in experiments 1–5 and the pooled data. Results from individual experiments indicate a trend towards a lower prevalence of infection in EVida3, which is only significant (*) for experiment 2 (<i>P</i> = 0.02) and the pooled data 1–5 (<i>P</i><0.001).</p>", "links"=>[], "tags"=>["prevalence", "infections", "transgenic", "strains"], "article_id"=>474872, "categories"=>["Biotechnology", "Genetics", "Infectious Diseases"], "users"=>["Janet M. Meredith", "Sanjay Basu", "Derric D. Nimmo", "Isabelle Larget-Thiery", "Emma L. Warr", "Ann Underhill", "Clare C. McArthur", "Victoria Carter", "Hilary Hurd", "Catherine Bourgouin", "Paul Eggleston"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0014587.g004", "stats"=>{"downloads"=>1, "page_views"=>5, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Parasite_intensity_and_prevalence_following_P_y_nigeriensis_infections_of_transgenic_strains_E_and_EVida3_/474872", "title"=>"Parasite intensity and prevalence following <i>P. y. nigeriensis</i> infections of transgenic strains E and EVida3.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-01-25 01:21:12"}
  • {"files"=>["https://ndownloader.figshare.com/files/804386"], "description"=>"<p>(A) Expression of Vida3 in strain EVida3 following a bloodmeal. (i) Representative gels showing semi-quantitative RT-PCR products of <i>rpL7a</i> (15 cycles, upper panel) and Vida3 (25 cycles, lower panel) from samples taken at 0, 1.5, 3, 6, 12, 24 and 48 hours post-bloodmeal. (ii) Mean relative intensity with standard errors of Vida3 PCR products. Following quantification, Vida3 RT-PCR bands were normalised to ribosomal protein <i>rpL7a</i> RT-PCR products from the same samples and expressed relative to the 0 time point. The histogram represents data from a minimum of two replicates. (B) RT-PCR of Vida3 expression in the midgut and carcass 6 hours post blood-meal.</p>", "links"=>[], "tags"=>["vida3"], "article_id"=>474745, "categories"=>["Biotechnology", "Genetics", "Infectious Diseases"], "users"=>["Janet M. Meredith", "Sanjay Basu", "Derric D. Nimmo", "Isabelle Larget-Thiery", "Emma L. Warr", "Ann Underhill", "Clare C. McArthur", "Victoria Carter", "Hilary Hurd", "Catherine Bourgouin", "Paul Eggleston"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0014587.g003", "stats"=>{"downloads"=>1, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Expression_of_Vida3_from_the_AgCP_promoter_/474745", "title"=>"Expression of Vida3 from the <i>AgCP</i> promoter.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-01-25 01:19:05"}
  • {"files"=>["https://ndownloader.figshare.com/files/804588"], "description"=>"<p>(A) Boxplots show parasite intensity for strains E (white boxes) and EVida3 (grey boxes) from 8 independent experiments. Vertical lines denote the 95% confidence interval, horizontal lines with symbol mark the median and the interquartile range of the data is boxed. Significant reductions in intensity (*) were observed for experiments 2 (EVida3; <i>P</i><0.0001), 4 (E; <i>P</i><0.0001), 5 (E; <i>P</i> = 0.0002), 7 (EVida3; <i>P</i><0.0001), 8 (EVida3; <i>P</i><0.0001). (B) Histograms show prevalence for strains E (white bars) and EVida3 (grey bars) in experiments 1–8. Significant reductions (*) were observed for experiments 2 (EVida3; <i>P</i> = 0.01), 3 (EVida3; <i>P</i> = 0.03) and 4 (E; <i>P</i> = 0.0003).</p>", "links"=>[], "tags"=>["prevalence", "infections", "transgenic", "strains"], "article_id"=>474940, "categories"=>["Biotechnology", "Genetics", "Infectious Diseases"], "users"=>["Janet M. Meredith", "Sanjay Basu", "Derric D. Nimmo", "Isabelle Larget-Thiery", "Emma L. Warr", "Ann Underhill", "Clare C. McArthur", "Victoria Carter", "Hilary Hurd", "Catherine Bourgouin", "Paul Eggleston"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0014587.g005", "stats"=>{"downloads"=>0, "page_views"=>1, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Parasite_intensity_and_prevalence_following_P_falciparum_infections_of_transgenic_strains_E_and_EVida3_/474940", "title"=>"Parasite intensity and prevalence following <i>P. falciparum</i> infections of transgenic strains E and EVida3.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-01-25 01:22:20"}
  • {"files"=>["https://ndownloader.figshare.com/files/401479", "https://ndownloader.figshare.com/files/401496", "https://ndownloader.figshare.com/files/401524", "https://ndownloader.figshare.com/files/401559"], "description"=>"<div><p>Diseases transmitted by mosquitoes have a devastating impact on global health and this is worsening due to difficulties with existing control measures and climate change. Genetically modified mosquitoes that are refractory to disease transmission are seen as having great potential in the delivery of novel control strategies. Historically the genetic modification of insects has relied upon transposable elements which have many limitations despite their successful use. To circumvent these limitations the <em>Streptomyces</em> phage phiC31 integrase system has been successfully adapted for site-specific transgene integration in insects. Here, we present the first site-specific transformation of <em>Anopheles gambiae</em>, the principal vector of human malaria. Mosquitoes were initially engineered to incorporate the phiC31 targeting site at a defined genomic location. A second phase of genetic modification then achieved site-specific integration of Vida3, a synthetic anti-malarial gene. Expression of Vida3, specifically in the midgut of bloodfed females, offered consistent and significant protection against <em>Plasmodium yoelii nigeriensis</em>, reducing average parasite intensity by 85%. Similar protection was observed against <em>Plasmodium falciparum</em> in some experiments, although protection was inconsistent. In the fight against malaria, it is imperative to establish a broad repertoire of both anti-malarial effector genes and tissue-specific promoters for their expression, enabling those offering maximum effect with minimum fitness cost to be identified. In the future, this technology will allow effective comparisons and informed choices to be made, potentially leading to complete transmission blockade.</p> </div>", "links"=>[], "tags"=>["site-specific", "anti-malarial", "transgenic", "reduces", "infections"], "article_id"=>139325, "categories"=>["Biotechnology", "Genetics", "Cancer"], "users"=>["Janet M. Meredith", "Sanjay Basu", "Derric D. Nimmo", "Isabelle Larget-Thiery", "Emma L. Warr", "Ann Underhill", "Clare C. McArthur", "Victoria Carter", "Hilary Hurd", "Catherine Bourgouin", "Paul Eggleston"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0014587.s001", "https://dx.doi.org/10.1371/journal.pone.0014587.s002", "https://dx.doi.org/10.1371/journal.pone.0014587.s003", "https://dx.doi.org/10.1371/journal.pone.0014587.s004"], "stats"=>{"downloads"=>28, "page_views"=>13, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Site_Specific_Integration_and_Expression_of_an_Anti_Malarial_Gene_in_Transgenic_Anopheles_gambiae_Significantly_Reduces_Plasmodium_Infections/139325", "title"=>"Site-Specific Integration and Expression of an Anti-Malarial Gene in Transgenic <em>Anopheles gambiae</em> Significantly Reduces <em>Plasmodium</em> Infections", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2011-01-25 02:35:25"}

PMC Usage Stats | Further Information

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