Name: measurement of lifespan in drosophila melanogaster
Uploaded: 2013-01-07T13:00:00
Description: Watch this Scientific Journal Video about Measurement of Lifespan in Drosophila melanogaster at JoVE.com

Dette arbejde blev støttet af midler fra Ellison Medical Foundation (SDP, <a href="http://www.ellisonfoundation.org/index.jsp" target="_blank">http://www.ellisonfoundation.org/index.jsp</a> ), NIH K01AG031917 (NJL, <a href="http://www.nih.gov" target="_blank">http://www.nih.gov/</a> ), NIH 5T32GM007315-35 (JR) og NIH R01AG030593 (SDP). Dette arbejde udnyttede ressourcerne i Drosophila Aging Core (DAC) af Nathan Shock Center of Excellence i Biologi af de aldrende finansieret af National Institute of Aging P30-AG-013.283 ( <a href="http://www.nih.gov" target="_blank">http://www.nih.gov/</a> ). Forfatterne vil gerne takke Pletcher Laboratorium for personer diskussioner og især Brian Chung for kritisk læsning af manuskriptet. Vi vil gerne anerkende Nick Asher og Kathryn Borowicz for assistance med indsamling af data.

Vi anbefaler opbevaring af eksperimentelle fødevarer, gær pasta, og drue agarplader, der vises i protokollen ved 4 ° C og bruge dem inden for 1-2 måneder, så længe mug og tørhed ikke har sat i. Standard miljøforhold for både larver og voksne fase involverer opretholdelse af fluer i en inkubator ved 25 ° C med en 12:12 timers lys mørke-cyklus og 60% relativ fugtighed. 1. Fremstilling af Eksperimentel Food <ol><li> For larve vækst, bruger vi en modificeret Caltech Medium 5,<...

<ol>
	<li>Toivonen, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=No+influence+of+Indy+on+lifespan+in+Drosophila+after+correction+for+genetic+and+cytoplasmic+background+effects.">No influence of Indy on lifespan in Drosophila after correction for genetic and cytoplasmic background effects.</a> PLoS Genet. 3, e95 (2007).</li><li>Spencer, C. C., Howell, C. E., Wright, A. R., Promislow, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Testing+an+'aging+gene'+in+long-lived+drosophila+strains:+increased+longevity+depends+on+sex+and+genetic+background.">Testing an 'aging gene' in long-lived drosophila strains: increased longevity depends on sex and genetic background.</a> Aging Cell. 2, 123-130 (2003).</li><li>Burnett, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absence+of+effects+of+Sir2+overexpression+on+lifespan+in+C.+elegans+and+Drosophila.">Absence of effects of Sir2 overexpression on lifespan in C. elegans and Drosophila.</a> Nature. 477, 482-485 (2011).</li><li>Bokov, A. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Does+reduced+IGF-1R+signaling+in+Igf1r+/-+mice+alter+aging?.">Does reduced IGF-1R signaling in Igf1r+/- mice alter aging?.</a> PLoS One. 6, e26891 (2011).</li><li>Lewis, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+standard+food+medium.">A new standard food medium.</a> Drosophila Information Service. 34, 117-118 (1960).</li><li>Skorupa, D. A., Dervisefendic, A., Zwiener, J., Pletcher, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dietary+composition+specifies+consumption,+obesity,+and+lifespan+in+Drosophila+melanogaster.">Dietary composition specifies consumption, obesity, and lifespan in Drosophila melanogaster.</a> Aging Cell. 7, 478-490 (2008).</li><li>Rera, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+longevity+and+tissue+homeostasis+by+the+Drosophila+PGC-1+homolog.">Modulation of longevity and tissue homeostasis by the Drosophila PGC-1 homolog.</a> Cell Metab. 14, 623-634 (2011).</li><li>Kaplan, E. L., Meier, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonparametric+Estimation+from+Incomplete+Observations.">Nonparametric Estimation from Incomplete Observations.</a> Journal of the American Statistical Association. 53, 457-481 (1958).</li><li>Pletcher, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitigating+the+Tithonus+Error:+Genetic+Analysis+of+Mortality+Phenotypes.">Mitigating the Tithonus Error: Genetic Analysis of Mortality Phenotypes.</a> Sci. Aging Knowl. Environ. 2002, pe14 (2002).</li><li>Pletcher, S. D., Khazaeli, A. A., Curtsinger, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+do+life+spans+differ?+Partitioning+mean+longevity+differences+in+terms+of+age-specific+mortality+parameters.">Why do life spans differ? Partitioning mean longevity differences in terms of age-specific mortality parameters.</a> J. Gerontol. A Biol. Sci. Med. Sci. 55, 381-389 (2000).</li><li>Promislow, , Tatar, , Pletcher, , Carey, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Below-threshold+mortality:+implications+for+studies+in+evolution,+ecology+and+demography.">Below-threshold mortality: implications for studies in evolution, ecology and demography.</a> Journal of Evolutionary Biology. 12, 314-328 (1999).</li><li>Pletcher, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Model+fitting+and+hypothesis+testing+for+age-specific+mortality+data.">Model fitting and hypothesis testing for age-specific mortality data.</a> Journal of Evolutionary Biology. 12, 430-439 (1999).</li><li>Partridge, L., Gems, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Benchmarks+for+ageing+studies.">Benchmarks for ageing studies.</a> Nature. 450, 165-167 (2007).</li><li>Roman, G., Endo, K., Zong, L., Davis, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P[Switch],+a+system+for+spatial+and+temporal+control+of+gene+expression+in+Drosophila+melanogaster.">P[Switch], a system for spatial and temporal control of gene expression in Drosophila melanogaster.</a> Proceedings of the National Academy of Sciences of the United States of America. 98, 12602-12607 (2001).</li><li>Ford, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alteration+of+Drosophila+life+span+using+conditional,+tissue-specific+expression+of+transgenes+triggered+by+doxycyline+or+RU486/Mifepristone.">Alteration of Drosophila life span using conditional, tissue-specific expression of transgenes triggered by doxycyline or RU486/Mifepristone.</a> Exp. Gerontol. 42, 483-497 (2007).</li><li>Priest, N. K., Mackowiak, B., Promislow, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+parental+age+effects+on+the+evolution+of+aging.">The role of parental age effects on the evolution of aging.</a> Evolution. 56, 927-935 (2002).</li><li>Smith, E. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feeding+Drosophila+a+biotin-deficient+diet+for+multiple+generations+increases+stress+resistance+and+lifespan+and+alters+gene+expression+and+histone+biotinylation+patterns.">Feeding Drosophila a biotin-deficient diet for multiple generations increases stress resistance and lifespan and alters gene expression and histone biotinylation patterns.</a> J. Nutr. 137, 2006-2012 (2007).</li><li>Sorensen, J. G., Loeschcke, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Larval+crowding+in+Drosophila+melanogaster+induces+Hsp70+expression,+and+leads+to+increased+adult+longevity+and+adult+thermal+stress+resistance.">Larval crowding in Drosophila melanogaster induces Hsp70 expression, and leads to increased adult longevity and adult thermal stress resistance.</a> J. Insect Physiol. 47, 1301-1307 (2001).</li><li>Bass, T. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+dietary+restriction+protocols+in+Drosophila.">Optimization of dietary restriction protocols in Drosophila.</a> J. Gerontol. A Biol. Sci. Med. Sci. 62, 1071-1081 (2007).</li><li>Miquel, J., Lundgren, P. R., Bensch, K. G., Atlan, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+temperature+on+the+life+span,+vitality+and+fine+structure+of+Drosophila+melanogaster.">Effects of temperature on the life span, vitality and fine structure of Drosophila melanogaster.</a> Mechanisms of Ageing and Development. 5, 347-370 (1976).</li><li>Pittendrigh, C. S., Minis, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+systems:+longevity+as+a+function+of+circadian+resonance+in+Drosophila+melanogaster.">Circadian systems: longevity as a function of circadian resonance in Drosophila melanogaster.</a> Proceedings of the National Academy of Sciences of the United States of America. 69, 1537-1539 (1972).</li><li>Joshi, A., Mueller, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+crowding+effects+on+longevity+in+Drosophila+melanogaster:+Increase+in+age-dependent+mortality.">Adult crowding effects on longevity in Drosophila melanogaster: Increase in age-dependent mortality.</a> Current Science. 72, 255-260 (1997).</li><li>Ja, W. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prandiology+of+Drosophila+and+the+CAFE+assay.">Prandiology of Drosophila and the CAFE assay.</a> Proceedings of the National Academy of Sciences of the United States of America. 104, 8253-8256 (2007).</li><li>Lee, K. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lifespan+and+reproduction+in+Drosophila:+New+insights+from+nutritional+geometry.">Lifespan and reproduction in Drosophila: New insights from nutritional geometry.</a> Proceedings of the National Academy of Sciences of the United States of America. 105, 2498-2503 (2008).</li><li>Gargano, J. W., Martin, I., Bhandari, P., Grotewiel, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+iterative+negative+geotaxis+(RING):+a+new+method+for+assessing+age-related+locomotor+decline+in+Drosophila.">Rapid iterative negative geotaxis (RING): a new method for assessing age-related locomotor decline in Drosophila.</a> Experimental gerontology. 40, 386-395 (2005).</li></ol>

Protokollen præsenteres her beskriver en fremgangsmåde til fremstilling af reproducerbare målinger af voksne levetid i Drosophila som er passende til vurdering af genetiske, farmakologiske, og miljømæssige tiltag. Afgørende aspekter af protokollen omfatter omhyggelig styring af larveudvikling miljø, minimere voksen stress, og minimerer skævhed tværs forsøgsgrupper og kontroller. Vi præsenterer også brugen af ​​dLife levetid eksperimentet management software. Ved blot at fastgøre en stregkode el...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer (valgfrit) </td></tr><tr><td> Tørgær </td><td> Fleishmann s Gær </td><td> 2192 </td><td></td></tr><tr><td> Grape Agar Powder Premix </td><td> Genesee Scientific </td><td> 47-102 </td><td></td></tr><tr><td> Store embryonindsamlingsteam Bure </td><td> Genesee Scientific </td><td> 59-101 </td><td></td></tr><tr><td> Store Udskiftning End Caps </td><td> Genesee Scientific </td><td> 59-103 </td><td></td></tr><tr><td> 6 ounce kvadratisk bund Flasker, polypropylen </td><td> Genesee Scientific </td><td> 32-130 </td><td></td></tr><tr><td> Flugs Låg til Stock Flasker </td><td> Genesee Scientific </td><td> 49-100 </td><td></td></tr><tr><td> DrosophilaVials, Wide, polystyren </td><td> Genesee Scientific </td><td> 32-117 </td><td></td></tr><tr><td> Flugs Låg til Brede Hætteglas </td><td> Genesee Scientific </td><td> 49-101 </td><td></td></tr><tr><td> Brede mundingsstørrelser Aardvark Pipettespidser, 200 ul </td><td> Denville Scientific </td><td> P1105-CP </td><td></td></tr><tr><td> Flystuff Flypad, Standard Size </td><td> Genesee Scientific </td><td> 59-114 </td><td></td></tr><tr><td> BD Falcon 15 ml koniske centrifugerør </td><td> Fisher Scientific </td><td> 14-959-70C </td><td></td></tr><tr><td> Fisherbrand petriskåle med klare låg, Raised Ridge, 100 OD x 15 mm H; </td><td> Fisher Scientific </td><td> 08-757-12 </td><td></td></tr><tr><td> Kimax * Colorware Kolber 1.000 ml gul </td><td> Fisher Scientific </td><td> 10-200-47 </td><td></td></tr><tr><td> PBS pH 7,4 10x </td><td> IVitrogen </td><td> 70011044 </td><td></td></tr><tr><td> Gelidium Agar </td><td> Mooragar </td><td> n / a </td><td></td></tr><tr><td> Ølgær </td><td> MP Biomedicals </td><td> 0290331280 </td><td></td></tr><tr><td> Sukker </td><td> Kroger </td><td> n / a </td><td></td></tr><tr><td> Tegosept </td><td> Genesee Scientific </td><td> 20-266 </td><td> Fly Food Konserveringsmiddel </td></tr><tr><td> Propionsyre, 99% </td><td> Acros Organics </td><td> 149300025 </td><td> Fly Food Konserveringsmiddel </td></tr><tr><td> Kanamycinsulfat </td><td> ISC BioExpress </td><td> 0408-10G </td><td></td></tr><tr><td> Tetracyklin HCI </td><td> VWR </td><td> 80058-724 </td><td></td></tr></tbody></table>

measurement of lifespan in drosophila melanogaster

Aldring er et fænomen, der resulterer i steady fysiologiske forringelse af næsten alle organismer i hvilke det er blevet undersøgt, hvilket fører til nedsat fysisk ydeevne og øget risiko for sygdom. Individuel aldring er åbenbart på populationsniveau som en stigning i aldersafhængig dødelighed, som ofte måles i laboratoriet ved at observere levetid i store kohorter af aldersmatchede individer. Eksperimenter, der søger at kvantificere det omfang, hvori genetiske eller miljømæssige manipulationer indvirkning levetid i simple model organismer har været bemærkelsesværdigt vellykkede til forståelse af aspekter ved aldring, som er konserveret på tværs af enheder samt for inspirerende nye strategier til at udvide levetiden og forhindre aldersbetinget sygdom hos pattedyr . Den eddike flyve, Drosophila melanogaster, er en attraktiv model organisme for at studere de mekanismer af aldring på grund af sin relativt korte levetid, praktisk dyrehold, og letkøbte genetik.Men demografiske foranstaltninger på ældning, herunder alder-specifik overlevelse og dødelighed, er overordentligt modtagelige for selv mindre variationer i eksperimentel design og miljø, og opretholdelse af strenge laboratoriepraksis for varigheden af ​​aldrende eksperimenter er påkrævet. Disse betragtninger, sammen med behovet for at praktisere omhyggelig kontrol af genetisk baggrund, er af afgørende betydning for at skabe robuste målinger. Faktisk er der mange bemærkelsesværdige kontroverser omkring slutning fra levealder forsøg i gær, orme, fluer og mus, der er blevet sporet til miljømæssige eller genetiske artefakter 1-4. I denne protokol, beskriver vi en række procedurer, der er blevet optimeret gennem mange års måling levetid i Drosophila hjælp laboratorium hætteglas. Vi beskriver også anvendelsen af dLife software, som blev udviklet af vort laboratorium og kan hentes ( <a href="http://sitemaker.umich.edu/pletcherlab/software">http://sitemaker.umich.edu/ple</a>tcherlab / software). dLife accelererer throughput og fremmer god praksis ved at indarbejde optimale eksperimenterende design, forenkle flyve håndtering og indsamling af data, og standardisere data analyse. Vi vil også diskutere de mange potentielle faldgruber i design, indsamling, og fortolkning af levetid data, og vi leverer skridt til at undgå disse farer.

En forenklet ordning af protokollen er vist i figur 1, hvor de vigtigste trin er skitseret. Synkroniseringen del af protokollen kan anvendes til forskellige assays, der kræver aldersmatchede voksne fluer. Typiske efterladte kurver for vildtype-fluer er vist i figur 2a, ved hjælp af dLife eksperimentet management software (figur 2b, c). Voksne hanner normalt lever kortere, med begge populationer opnå en middelværdi og median levetid på&g...

Watch this Scientific Journal Video about Measurement of Lifespan in Drosophila melanogaster at JoVE.com

Measurement of Lifespan in Drosophila melanogaster

Drosophila melanogaster er en kraftfuld model organisme for at udforske den molekylære basis for levetiden regulering. Denne protokol vil diskutere de skridt, der er involveret i at generere en reproducerbar, befolkning-baseret måling af levetiden samt potentielle faldgruber og hvordan man undgår dem.

Måling af Levetid i<em&gt; Drosophila melanogaster</em

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced ...

Research

JoVE Journal

Biology

Measurement of Lifespan in Drosophila melanogaster

Dissection of Larval CNS in Drosophila Melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field.  Studying ...

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained  and  ...

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

Dissection of Oenocytes from Adult Drosophila melanogaster

Dissection of Oenocytes from Adult Drosophila melanogaster

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides ...

Isolation of Drosophila melanogaster Testes

Isolation of Drosophila melanogaster Testes

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline ...

Imaging Through the Pupal Case of Drosophila melanogaster

Imaging Through the Pupal Case of Drosophila melanogaster

Imaging Gennem puppe Case of<em&gt; Drosophila melanogaster</em

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Measurement of Metabolic Rate in Drosophila using Respirometry

Måling af stofskifte i<em&gt; Drosophila</em&gt; Hjælp respirometri

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster

Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster

Forbedret Northern Blot detektering af små RNA-art i<em&gt; Drosophila melanogaster</em

The last decades have witnessed the explosion of scientific interest around gene expression control mechanisms at the RNA level. This branch of molecular ...

Maintenance of a Drosophila melanogaster Population Cage

Maintenance of a Drosophila melanogaster Population Cage

Vedligeholdelse af en<em&gt; Drosophila melanogaster</em&gt; Befolkning Cage

Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life ...