{"corpus_id":9350429,"paper_sha":"21c2624fa22d531b9fb1396d8e78765dd0fa3d2c","doi":"10.1371/journal.pone.0040075","arxiv_id":null,"pmid":22768223,"pmcid":"3386905","mag_id":2000418604,"dblp_id":null,"acl_id":null,"title":"piggyBac Transposon-Mediated Transgenesis in the Apicomplexan Parasite Eimeria tenella","year":2012,"publication_date":"2012-06-29","venue":"PLoS ONE","journal":{"name":"PLoS ONE","pages":null,"volume":"7"},"journal_issn":null,"journal_title":null,"publication_types":["JournalArticle"],"pubmed_pub_types":["Journal Article","Research Support, Non-U.S. Gov't"],"s2_fields_of_study":["Biology","Medicine"],"reference_count":27,"citation_count":17,"influential_citation_count":2,"is_open_access":true,"arxiv_categories":null,"arxiv_license":null,"arxiv_journal_ref":null,"mesh_headings":[{"d":"Animals","mj":false,"ui":"D000818"},{"d":"Animals, Genetically Modified","mj":false,"ui":"D030801"},{"d":"Bacterial Proteins","mj":false,"qs":[{"q":"metabolism","mj":false,"ui":"Q000378"}],"ui":"D001426"},{"d":"Base Sequence","mj":false,"ui":"D001483"},{"d":"DNA Transposable Elements","mj":false,"qs":[{"q":"genetics","mj":true,"ui":"Q000235"}],"ui":"D004251"},{"d":"Eimeria tenella","mj":false,"qs":[{"q":"cytology","mj":false,"ui":"Q000166"},{"q":"genetics","mj":true,"ui":"Q000235"}],"ui":"D016786"},{"d":"Flow Cytometry","mj":false,"ui":"D005434"},{"d":"Gene Transfer Techniques","mj":true,"ui":"D018014"},{"d":"Genome, Protozoan","mj":false,"qs":[{"q":"genetics","mj":false,"ui":"Q000235"}],"ui":"D018503"},{"d":"Luminescent Proteins","mj":false,"qs":[{"q":"metabolism","mj":false,"ui":"Q000378"}],"ui":"D008164"},{"d":"Molecular Sequence Data","mj":false,"ui":"D008969"},{"d":"Mutagenesis, Insertional","mj":false,"qs":[{"q":"genetics","mj":false,"ui":"Q000235"}],"ui":"D016254"},{"d":"Oocysts","mj":false,"qs":[{"q":"cytology","mj":false,"ui":"Q000166"},{"q":"metabolism","mj":false,"ui":"Q000378"}],"ui":"D033661"},{"d":"Parasites","mj":false,"qs":[{"q":"cytology","mj":false,"ui":"Q000166"},{"q":"genetics","mj":true,"ui":"Q000235"}],"ui":"D010271"},{"d":"Plasmids","mj":false,"qs":[{"q":"genetics","mj":false,"ui":"Q000235"}],"ui":"D010957"},{"d":"Reproducibility of Results","mj":false,"ui":"D015203"}],"chemicals":[{"n":"Bacterial Proteins","ui":"D001426","reg":"0"},{"n":"DNA Transposable Elements","ui":"D004251","reg":"0"},{"n":"Luminescent Proteins","ui":"D008164","reg":"0"},{"n":"yellow fluorescent protein, Bacteria","ui":"C054562","reg":"0"}],"comments_corrections":null,"source_flags":5,"s2_open_access_pdf_url":"https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0040075&type=printable","s2_open_access_landing_url":"https://www.semanticscholar.org/paper/21c2624fa22d531b9fb1396d8e78765dd0fa3d2c","s2_open_access_license":"CCBY","s2_open_access_status":"GOLD","pmc_open_access_pdf_url":null,"pmc_open_access_landing_url":null,"pmc_open_access_license":null,"pmc_open_access_status":null,"unpaywall_open_access_pdf_url":null,"unpaywall_open_access_landing_url":null,"unpaywall_open_access_license":null,"unpaywall_open_access_status":null,"abstract":"piggyBac, a type II transposon that is useful for efficient transgenesis and insertional mutagenesis, has been used for effective and stable transfection in a wide variety of organisms. In this study we investigate the potential use of the piggyBac transposon system for forward genetics studies in the apicomplexan parasite Eimeria tenella. Using the restriction enzyme-mediated integration (REMI) method, E. tenella sporozoites were electroporated with a donor plasmid containing the enhanced yellow fluorescent protein (EYFP) gene flanked by piggyBac inverted terminal repeats (ITRs), an Asc I-linearized helper plasmid containing the transposase gene and the restriction enzyme Asc I. Subsequently, electroporated sporozoites were inoculated into chickens via the cloacal route and transfected progeny oocysts expressing EYFP were sorted by flow cytometry. A transgenic E. tenella population was selected by successive in vivo passage. Southern-blotting analysis showed that exogenous DNA containing the EYFP gene was integrated into the parasite genome at a limited number of integration sites and that the inserted part of the donor plasmid was the fragment located between the 5′ and 3′ ITRs as indicated by primer-specific PCR screening. Genome walking revealed that the insertion sites were TTAA-specific, which is consistent with the transposition characteristics of piggyBac.","claims":[{"public_id":"cl_6d53c06a69b39257fe451e7d9a2c8f3d","status":"active","text":"Electroporated sporozoites carrying an EYFP donor plasmid and a transposase helper plasmid produced transfected progeny oocysts that could be sorted by flow cytometry and selected through successive in vivo passage.","confidence":0.93,"contributors":[{"id":1,"public_id":"12632b8b5f","public_label":"Anonymous (12632b8b5f)","roles":["extraction"],"url":"https://sah.borca.ai/u/12632b8b5f"}],"url":"https://sah.borca.ai/claims/cl_6d53c06a69b39257fe451e7d9a2c8f3d"},{"public_id":"cl_0ddcac91c273dea3728b290acd88ac87","status":"active","text":"Exogenous EYFP-containing DNA integrated into the parasite genome at a limited number of sites, with the inserted sequence corresponding to the region between the 5′ and 3′ piggyBac inverted terminal repeats.","confidence":0.95,"contributors":[{"id":1,"public_id":"12632b8b5f","public_label":"Anonymous (12632b8b5f)","roles":["extraction"],"url":"https://sah.borca.ai/u/12632b8b5f"}],"url":"https://sah.borca.ai/claims/cl_0ddcac91c273dea3728b290acd88ac87"},{"public_id":"cl_6ba82eda6026be2ee6949f9fc7fd85db","status":"active","text":"Insertion sites were TTAA-specific, matching the expected transposition characteristics of piggyBac.","confidence":0.96,"contributors":[{"id":1,"public_id":"12632b8b5f","public_label":"Anonymous (12632b8b5f)","roles":["extraction"],"url":"https://sah.borca.ai/u/12632b8b5f"}],"url":"https://sah.borca.ai/claims/cl_6ba82eda6026be2ee6949f9fc7fd85db"},{"public_id":"cl_072c3ec21dae874febf619921f56bbbb","status":"active","text":"piggyBac transposon-mediated transgenesis is feasible in Eimeria tenella for forward genetics applications.","confidence":0.97,"contributors":[{"id":1,"public_id":"12632b8b5f","public_label":"Anonymous (12632b8b5f)","roles":["extraction"],"url":"https://sah.borca.ai/u/12632b8b5f"}],"url":"https://sah.borca.ai/claims/cl_072c3ec21dae874febf619921f56bbbb"}],"concepts":[{"public_id":"co_37ec8ae77a6a37c643e7a1eaabd0eec8","status":"active","name":"TTAA-specific insertion sites","description":"Genomic target sites characterized by the TTAA sequence motif preferred by piggyBac insertion.","types":["sequence motif","insertion site"],"aliases":["TTAA sites"],"contributors":[{"id":1,"public_id":"12632b8b5f","public_label":"Anonymous (12632b8b5f)","roles":["extraction"],"url":"https://sah.borca.ai/u/12632b8b5f"}],"url":"https://sah.borca.ai/concepts/co_37ec8ae77a6a37c643e7a1eaabd0eec8"},{"public_id":"co_400fdbe6e98ffda1ae0fb693a4a186ff","status":"active","name":"enhanced yellow fluorescent protein","description":"A fluorescent reporter gene used to mark transfected parasite cells.","types":["reporter 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site.","types":["method"],"aliases":[],"contributors":[{"id":1,"public_id":"12632b8b5f","public_label":"Anonymous (12632b8b5f)","roles":["extraction"],"url":"https://sah.borca.ai/u/12632b8b5f"}],"url":"https://sah.borca.ai/concepts/co_4fcf3f83bb8ac477c226a40835ceaf18"},{"public_id":"co_57b0e0fc0931335c9c7712bda637fa99","status":"active","name":"forward genetics","description":"A genetics strategy that uses induced mutations or insertions to identify gene function.","types":["research approach"],"aliases":[],"contributors":[{"id":1,"public_id":"12632b8b5f","public_label":"Anonymous (12632b8b5f)","roles":["extraction"],"url":"https://sah.borca.ai/u/12632b8b5f"}],"url":"https://sah.borca.ai/concepts/co_57b0e0fc0931335c9c7712bda637fa99"},{"public_id":"co_6162e12a0dc3354428de56675dd3d75c","status":"active","name":"restriction enzyme-mediated integration","description":"A transfection-assisted method used here to introduce plasmid DNA into sporozoites.","types":["method"],"aliases":["REMI"],"contributors":[{"id":1,"public_id":"12632b8b5f","public_label":"Anonymous (12632b8b5f)","roles":["extraction"],"url":"https://sah.borca.ai/u/12632b8b5f"}],"url":"https://sah.borca.ai/concepts/co_6162e12a0dc3354428de56675dd3d75c"},{"public_id":"co_69dfb74c41558e79016074554b7b3cf0","status":"active","name":"flow cytometry","description":"A cell-sorting technique used to isolate oocysts expressing the fluorescent reporter.","types":["measurement technique"],"aliases":[],"contributors":[{"id":1,"public_id":"12632b8b5f","public_label":"Anonymous (12632b8b5f)","roles":["extraction"],"url":"https://sah.borca.ai/u/12632b8b5f"}],"url":"https://sah.borca.ai/concepts/co_69dfb74c41558e79016074554b7b3cf0"},{"public_id":"co_72ddf968d0511c3ac1e5c0a38c383109","status":"active","name":"piggyBac inverted terminal repeats","description":"The terminal DNA repeat sequences that flank the donor construct and enable piggyBac 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