Establishment of transient and stable transfection systems for Babesia ovata
Bovine babesiosis is a tick-borne disease caused by several species of Babesia which produce acute and fatal disease in cattle and affect livestock industry worldwide. Babesia ovata is a benign species widespread in east Asian countries and causes anemia, particularly in cattle which are co-infected...
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creator | Hakimi, Hassan Yamagishi, Junya Kegawa, Yuto Kaneko, Osamu Kawazu, Shin-Ichiro Asada, Masahito |
description | Bovine babesiosis is a tick-borne disease caused by several species of Babesia which produce acute and fatal disease in cattle and affect livestock industry worldwide. Babesia ovata is a benign species widespread in east Asian countries and causes anemia, particularly in cattle which are co-infected with Theileria orientalis. The development of genetic manipulation methods is necessary to improve our understanding of the basic biology of protozoan pathogens toward a better control of disease. Such tools have not been developed for B. ovata, and are the aim of this study.
In this study we transfected constructs that were designed to evaluate the ability of several B. ovata promoter candidates to drive expression of a reporter luciferase. We found that the elongation factor-1 alpha intergenic region (ef-1α IG) and the actin 5' non-coding region (NR) had highest promoter activities. To establish a stable transfection system, we generated a plasmid construct in which the ef-1α IG promoter drives gfp expression, and the actin 5' NR mediates expression of the selectable marker hdhfr. The plasmid was designed for episomal transfection, as well as to integrate by double cross-over homologous recombination into the ef-1α locus. Circular or linearized plasmid was transfected by electroporation into in vitro cultured B. ovata and retention of the plasmid was facilitated by drug selection with 5 nM WR99210 initiated 48 h after transfection.
After one-week cultivation with WR99210, GFP-expressing parasites were observed by fluorescence microscopy. Integration of the plasmid construct into the ef-1α locus was confirmed by PCR, Southern blot analysis, and sequencing of recombination sites. These results confirm successful development of a stable transfection system for B. ovata.
The current study provides a fundamental molecular tool to aid in molecular and cellular studies of B. ovata. |
doi_str_mv | 10.1186/s13071-016-1439-z |
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In this study we transfected constructs that were designed to evaluate the ability of several B. ovata promoter candidates to drive expression of a reporter luciferase. We found that the elongation factor-1 alpha intergenic region (ef-1α IG) and the actin 5' non-coding region (NR) had highest promoter activities. To establish a stable transfection system, we generated a plasmid construct in which the ef-1α IG promoter drives gfp expression, and the actin 5' NR mediates expression of the selectable marker hdhfr. The plasmid was designed for episomal transfection, as well as to integrate by double cross-over homologous recombination into the ef-1α locus. Circular or linearized plasmid was transfected by electroporation into in vitro cultured B. ovata and retention of the plasmid was facilitated by drug selection with 5 nM WR99210 initiated 48 h after transfection.
After one-week cultivation with WR99210, GFP-expressing parasites were observed by fluorescence microscopy. Integration of the plasmid construct into the ef-1α locus was confirmed by PCR, Southern blot analysis, and sequencing of recombination sites. These results confirm successful development of a stable transfection system for B. ovata.
The current study provides a fundamental molecular tool to aid in molecular and cellular studies of B. ovata.</description><identifier>ISSN: 1756-3305</identifier><identifier>EISSN: 1756-3305</identifier><identifier>DOI: 10.1186/s13071-016-1439-z</identifier><identifier>PMID: 27008652</identifier><language>eng</language><publisher>England: BioMed Central</publisher><subject>actin ; anemia ; Artificial Gene Fusion ; Babesia ; Babesia - genetics ; babesiosis ; cattle ; cattle diseases ; disease control ; drugs ; East Asia ; electroporation ; fluorescence microscopy ; gene expression ; Gene Expression Profiling ; Gene Targeting ; Genes, Reporter ; genetic engineering ; Genetic Vectors ; Genetics, Microbial - methods ; Homologous Recombination ; in vitro culture ; intergenic DNA ; livestock and meat industry ; loci ; luciferase ; mixed infection ; parasites ; pathogens ; Plasmids ; polymerase chain reaction ; Promoter Regions, Genetic ; Protozoa ; Southern blotting ; Theileria orientalis ; transfection ; Transfection - methods</subject><ispartof>Parasites & vectors, 2016-03, Vol.9 (1), p.171-171, Article 171</ispartof><rights>Copyright BioMed Central 2016</rights><rights>Hakimi et al. 2016</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c504t-e7271852ea16d3df27d50ab9f8714adbc6038e0f1864a330eba9b9d493d031743</citedby><cites>FETCH-LOGICAL-c504t-e7271852ea16d3df27d50ab9f8714adbc6038e0f1864a330eba9b9d493d031743</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4806448/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4806448/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,860,881,27901,27902,53766,53768</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/27008652$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Hakimi, Hassan</creatorcontrib><creatorcontrib>Yamagishi, Junya</creatorcontrib><creatorcontrib>Kegawa, Yuto</creatorcontrib><creatorcontrib>Kaneko, Osamu</creatorcontrib><creatorcontrib>Kawazu, Shin-Ichiro</creatorcontrib><creatorcontrib>Asada, Masahito</creatorcontrib><title>Establishment of transient and stable transfection systems for Babesia ovata</title><title>Parasites & vectors</title><addtitle>Parasit Vectors</addtitle><description>Bovine babesiosis is a tick-borne disease caused by several species of Babesia which produce acute and fatal disease in cattle and affect livestock industry worldwide. Babesia ovata is a benign species widespread in east Asian countries and causes anemia, particularly in cattle which are co-infected with Theileria orientalis. The development of genetic manipulation methods is necessary to improve our understanding of the basic biology of protozoan pathogens toward a better control of disease. Such tools have not been developed for B. ovata, and are the aim of this study.
In this study we transfected constructs that were designed to evaluate the ability of several B. ovata promoter candidates to drive expression of a reporter luciferase. We found that the elongation factor-1 alpha intergenic region (ef-1α IG) and the actin 5' non-coding region (NR) had highest promoter activities. To establish a stable transfection system, we generated a plasmid construct in which the ef-1α IG promoter drives gfp expression, and the actin 5' NR mediates expression of the selectable marker hdhfr. The plasmid was designed for episomal transfection, as well as to integrate by double cross-over homologous recombination into the ef-1α locus. Circular or linearized plasmid was transfected by electroporation into in vitro cultured B. ovata and retention of the plasmid was facilitated by drug selection with 5 nM WR99210 initiated 48 h after transfection.
After one-week cultivation with WR99210, GFP-expressing parasites were observed by fluorescence microscopy. Integration of the plasmid construct into the ef-1α locus was confirmed by PCR, Southern blot analysis, and sequencing of recombination sites. These results confirm successful development of a stable transfection system for B. ovata.
The current study provides a fundamental molecular tool to aid in molecular and cellular studies of B. ovata.</description><subject>actin</subject><subject>anemia</subject><subject>Artificial Gene Fusion</subject><subject>Babesia</subject><subject>Babesia - genetics</subject><subject>babesiosis</subject><subject>cattle</subject><subject>cattle diseases</subject><subject>disease control</subject><subject>drugs</subject><subject>East Asia</subject><subject>electroporation</subject><subject>fluorescence microscopy</subject><subject>gene expression</subject><subject>Gene Expression Profiling</subject><subject>Gene Targeting</subject><subject>Genes, Reporter</subject><subject>genetic engineering</subject><subject>Genetic Vectors</subject><subject>Genetics, Microbial - methods</subject><subject>Homologous Recombination</subject><subject>in vitro culture</subject><subject>intergenic DNA</subject><subject>livestock and meat industry</subject><subject>loci</subject><subject>luciferase</subject><subject>mixed infection</subject><subject>parasites</subject><subject>pathogens</subject><subject>Plasmids</subject><subject>polymerase chain reaction</subject><subject>Promoter Regions, Genetic</subject><subject>Protozoa</subject><subject>Southern blotting</subject><subject>Theileria orientalis</subject><subject>transfection</subject><subject>Transfection - methods</subject><issn>1756-3305</issn><issn>1756-3305</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2016</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>BENPR</sourceid><recordid>eNqNUU1LxDAQDaL4sfoDvEjBi5fqTJMm6UVQ8QsWvOg5TNtUK22jSVdYf71ZdxX1JDlMZubN4808xvYRjhG1PAnIQWEKKFMUvEjf19g2qlymnEO-_uO_xXZCeAaQUORyk21lCkDLPNtm08swUtm14am3w5i4Jhk9DaFdJDTUyWfXLouNrcbWDUmYh9H2IWmcT86ptKGlxL3RSLtso6Eu2L1VnLCHq8v7i5t0end9e3E2TascxJhalSnUeWYJZc3rJlN1DlQWjVYoqC4rCVxbaOKKgqJ8W1JRFrUoeA0cleATdrrkfZmVva2rKNZTZ15825OfG0et-d0Z2ifz6N6M0CCF0JHgaEXg3evMhtH0bahs19Fg3SwY1EJyLlD9A6pUvCrCp6zDP9BnN_NDvEREFfHpHBcoXKIq70LwtvnWjWAWtpqlrSbaaha2mvc4c_Bz4e-JLx_5Bzsjnlw</recordid><startdate>20160323</startdate><enddate>20160323</enddate><creator>Hakimi, Hassan</creator><creator>Yamagishi, Junya</creator><creator>Kegawa, Yuto</creator><creator>Kaneko, Osamu</creator><creator>Kawazu, Shin-Ichiro</creator><creator>Asada, Masahito</creator><general>BioMed Central</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>3V.</scope><scope>7SN</scope><scope>7SS</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BENPR</scope><scope>C1K</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>F1W</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>H95</scope><scope>K9.</scope><scope>L.G</scope><scope>M0S</scope><scope>M1P</scope><scope>M7N</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>7X8</scope><scope>7S9</scope><scope>L.6</scope><scope>5PM</scope></search><sort><creationdate>20160323</creationdate><title>Establishment of transient and stable transfection systems for Babesia ovata</title><author>Hakimi, Hassan ; Yamagishi, Junya ; Kegawa, Yuto ; Kaneko, Osamu ; Kawazu, Shin-Ichiro ; Asada, Masahito</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c504t-e7271852ea16d3df27d50ab9f8714adbc6038e0f1864a330eba9b9d493d031743</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2016</creationdate><topic>actin</topic><topic>anemia</topic><topic>Artificial Gene Fusion</topic><topic>Babesia</topic><topic>Babesia - genetics</topic><topic>babesiosis</topic><topic>cattle</topic><topic>cattle diseases</topic><topic>disease control</topic><topic>drugs</topic><topic>East Asia</topic><topic>electroporation</topic><topic>fluorescence microscopy</topic><topic>gene expression</topic><topic>Gene Expression Profiling</topic><topic>Gene Targeting</topic><topic>Genes, Reporter</topic><topic>genetic engineering</topic><topic>Genetic Vectors</topic><topic>Genetics, Microbial - methods</topic><topic>Homologous Recombination</topic><topic>in vitro culture</topic><topic>intergenic DNA</topic><topic>livestock and meat industry</topic><topic>loci</topic><topic>luciferase</topic><topic>mixed infection</topic><topic>parasites</topic><topic>pathogens</topic><topic>Plasmids</topic><topic>polymerase chain reaction</topic><topic>Promoter Regions, Genetic</topic><topic>Protozoa</topic><topic>Southern blotting</topic><topic>Theileria orientalis</topic><topic>transfection</topic><topic>Transfection - methods</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Hakimi, Hassan</creatorcontrib><creatorcontrib>Yamagishi, Junya</creatorcontrib><creatorcontrib>Kegawa, Yuto</creatorcontrib><creatorcontrib>Kaneko, Osamu</creatorcontrib><creatorcontrib>Kawazu, Shin-Ichiro</creatorcontrib><creatorcontrib>Asada, Masahito</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>ProQuest Central (Corporate)</collection><collection>Ecology Abstracts</collection><collection>Entomology Abstracts (Full archive)</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Medical Database (Alumni Edition)</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest Central UK/Ireland</collection><collection>ProQuest Central Essentials</collection><collection>ProQuest Central</collection><collection>Environmental Sciences and Pollution Management</collection><collection>ProQuest One Community College</collection><collection>ProQuest Central Korea</collection><collection>ASFA: Aquatic Sciences and Fisheries Abstracts</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>Aquatic Science & Fisheries Abstracts (ASFA) 1: Biological Sciences & Living Resources</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>Aquatic Science & Fisheries Abstracts (ASFA) Professional</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Publicly Available Content Database</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>ProQuest Central China</collection><collection>MEDLINE - Academic</collection><collection>AGRICOLA</collection><collection>AGRICOLA - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Parasites & vectors</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Hakimi, Hassan</au><au>Yamagishi, Junya</au><au>Kegawa, Yuto</au><au>Kaneko, Osamu</au><au>Kawazu, Shin-Ichiro</au><au>Asada, Masahito</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Establishment of transient and stable transfection systems for Babesia ovata</atitle><jtitle>Parasites & vectors</jtitle><addtitle>Parasit Vectors</addtitle><date>2016-03-23</date><risdate>2016</risdate><volume>9</volume><issue>1</issue><spage>171</spage><epage>171</epage><pages>171-171</pages><artnum>171</artnum><issn>1756-3305</issn><eissn>1756-3305</eissn><abstract>Bovine babesiosis is a tick-borne disease caused by several species of Babesia which produce acute and fatal disease in cattle and affect livestock industry worldwide. Babesia ovata is a benign species widespread in east Asian countries and causes anemia, particularly in cattle which are co-infected with Theileria orientalis. The development of genetic manipulation methods is necessary to improve our understanding of the basic biology of protozoan pathogens toward a better control of disease. Such tools have not been developed for B. ovata, and are the aim of this study.
In this study we transfected constructs that were designed to evaluate the ability of several B. ovata promoter candidates to drive expression of a reporter luciferase. We found that the elongation factor-1 alpha intergenic region (ef-1α IG) and the actin 5' non-coding region (NR) had highest promoter activities. To establish a stable transfection system, we generated a plasmid construct in which the ef-1α IG promoter drives gfp expression, and the actin 5' NR mediates expression of the selectable marker hdhfr. The plasmid was designed for episomal transfection, as well as to integrate by double cross-over homologous recombination into the ef-1α locus. Circular or linearized plasmid was transfected by electroporation into in vitro cultured B. ovata and retention of the plasmid was facilitated by drug selection with 5 nM WR99210 initiated 48 h after transfection.
After one-week cultivation with WR99210, GFP-expressing parasites were observed by fluorescence microscopy. Integration of the plasmid construct into the ef-1α locus was confirmed by PCR, Southern blot analysis, and sequencing of recombination sites. These results confirm successful development of a stable transfection system for B. ovata.
The current study provides a fundamental molecular tool to aid in molecular and cellular studies of B. ovata.</abstract><cop>England</cop><pub>BioMed Central</pub><pmid>27008652</pmid><doi>10.1186/s13071-016-1439-z</doi><tpages>1</tpages><oa>free_for_read</oa></addata></record> |
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subjects | actin anemia Artificial Gene Fusion Babesia Babesia - genetics babesiosis cattle cattle diseases disease control drugs East Asia electroporation fluorescence microscopy gene expression Gene Expression Profiling Gene Targeting Genes, Reporter genetic engineering Genetic Vectors Genetics, Microbial - methods Homologous Recombination in vitro culture intergenic DNA livestock and meat industry loci luciferase mixed infection parasites pathogens Plasmids polymerase chain reaction Promoter Regions, Genetic Protozoa Southern blotting Theileria orientalis transfection Transfection - methods |
title | Establishment of transient and stable transfection systems for Babesia ovata |
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