Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

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dc.contributor.authorTimoshevskiy, Vladimir A.en
dc.contributor.authorSharma, Atashien
dc.contributor.authorSharakhov, Igor V.en
dc.contributor.authorSharakhova, Maria V.en
dc.contributor.departmentEntomologyen
dc.date.accessioned2017-01-05T23:41:00Zen
dc.date.available2017-01-05T23:41:00Zen
dc.date.issued2012-09-01en
dc.description.abstractFluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. One important application of this method is the development of high-quality physical maps useful for improving the genome assemblies for various organisms. The natural banding pattern of polytene and mitotic chromosomes provides guidance for the precise ordering and orientation of the genomic supercontigs. Among the three mosquito genera, namely Anopheles, Aedes, and Culex, a well-established chromosome-based mapping technique has been developed only for Anopheles, whose members possess readable polytene chromosomes. As a result of genome mapping efforts, 88% of the An. gambiae genome has been placed to precise chromosome positions. Two other mosquito genera, Aedes and Culex, have poorly polytenized chromosomes because of significant overrepresentation of transposable elements in their genomes. Only 31 and 9% of the genomic supercontings have been assigned without order or orientation to chromosomes of Ae. aegypti and Cx. quinquefasciatus, respectively. Mitotic chromosome preparation for these two species had previously been limited to brain ganglia and cell lines. However, chromosome slides prepared from the brain ganglia of mosquitoes usually contain low numbers of metaphase plates. Also, although a FISH technique has been developed for mitotic chromosomes from a cell line of Ae. aegypti, the accumulation of multiple chromosomal rearrangements in cell line chromosomes makes them useless for genome mapping. Here we describe a simple, robust technique for obtaining high-quality mitotic chromosome preparations from imaginal discs (IDs) of 4th instar larvae which can be used for all three genera of mosquitoes. A standard FISH protocol is optimized for using BAC clones of genomic DNA as a probe on mitotic chromosomes of Ae. aegypti and Cx. quinquefasciatus, and for utilizing an intergenic spacer (IGS) region of ribosomal DNA (rDNA) as a probe on An. gambiae chromosomes. In addition to physical mapping, the developed technique can be applied to population cytogenetics and chromosome taxonomy/systematics of mosquitoes and other insect groups.en
dc.description.versionPublished versionen
dc.format.extent? - ? (7) page(s)en
dc.format.mimetypeapplication/pdfen
dc.identifier.doihttps://doi.org/10.3791/4215en
dc.identifier.issn1940-087Xen
dc.identifier.issue67en
dc.identifier.urihttp://hdl.handle.net/10919/73973en
dc.language.isoenen
dc.publisherJournal of Visualized Experimentsen
dc.relation.urihttp://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcApp=PARTNER_APP&SrcAuth=LinksAMR&KeyUT=WOS:000209225500032&DestLinkType=FullRecord&DestApp=ALL_WOS&UsrCustomerID=930d57c9ac61a043676db62af60056c1en
dc.rightsCreative Commons Attribution-NonCommercial-NoDerivs 3.0 Unporteden
dc.rights.urihttp://creativecommons.org/licenses/by-nc-nd/3.0/en
dc.subjectimmunologyen
dc.subjectissue 67en
dc.subjectgeneticsen
dc.subjectmolecular biologyen
dc.subjectentomologyen
dc.subjectinfectious diseaseen
dc.subjectimaginal discsen
dc.subjectmitotic chromosomesen
dc.subjectgenome mappingen
dc.subjectfishen
dc.subjectfluorescent in situ hybridizationen
dc.subjectmosquitoesen
dc.subjectanophelesen
dc.subjectaedesen
dc.subjectculexen
dc.titleFluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoesen
dc.title.serialJove-Journal of Visualized Experimentsen
dc.typeArticle - Refereeden
dc.type.dcmitypeTexten
pubs.organisational-group/Virginia Techen
pubs.organisational-group/Virginia Tech/Agriculture & Life Sciencesen
pubs.organisational-group/Virginia Tech/Agriculture & Life Sciences/CALS T&R Facultyen
pubs.organisational-group/Virginia Tech/Agriculture & Life Sciences/Entomologyen
pubs.organisational-group/Virginia Tech/All T&R Facultyen
pubs.organisational-group/Virginia Tech/Faculty of Health Sciencesen

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