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

Detta arbete stöddes av medel från 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) och NIH R01AG030593 (SDP). Detta arbete utnyttjade resurserna i Drosophila Aging Core (DAC) i Nathan Shock Center of Excellence i biologi av åldrandet finansieras av National Institute of Aging P30-AG-013.283 ( <a href="http://www.nih.gov" target="_blank">http://www.nih.gov/</a> ). Författarna vill tacka Pletcher Laboratoriet för hjälp diskussioner och särskilt Brian Chung för kritisk läsning av manuskriptet. Vi vill tacka för Nick Asher och Kathryn Borowicz för hjälp med datainsamling.

Vi rekommenderar att lagra experimentella livsmedel, jäst pasta och druva agarplattor som visas i protokollet vid 4 ° C och använda dem inom 1-2 månader, så länge mögel och torrhet inte har ställt in standard miljöförhållanden för både larver och vuxna steg inbegripa bibehållande av flugor i en inkubator vid 25 ° C med en 12:12 timmars ljus mörkercykel och 60% relativ fuktighet. 1. Beredning av experimentell mat <ol><li> För larver tillväxt använder vi en modifierad Calt...

<ol>
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Protokollet som presenteras här beskriver ett förfarande för framställning av reproducerbara mätningar av vuxen livslängd i Drosophila som är anpassningsbart för bedömning av genetiska, farmakologiska och miljömässiga åtgärder. Avgörande aspekterna av protokollet inkluderar noggrant kontrollera larver utvecklingsmiljö, minimera vuxna stress och minimera partiskhet över experimentella grupper och kontroller. Vi presenterar också användningen av dLife livslängd experimentet programvara. Genom a...

<table border="1"><tbody><tr><td> Namnet av reagenset </td><td> Företag </td><td> Katalognummer </td><td> Kommentarer (tillval) </td></tr><tr><td> Aktiv torrjäst </td><td> Fleishmann jäst </td><td> 2192 </td><td></td></tr><tr><td> Grape agarpulver Premix </td><td> Genesee Scientific </td><td> 47-102 </td><td></td></tr><tr><td> Stora embryosamlingsgrupp Burar </td><td> Genesee Scientific </td><td> 59-101 </td><td></td></tr><tr><td> Stora Replacement Gavlar </td><td> Genesee Scientific </td><td> 59-103 </td><td></td></tr><tr><td> 6 oz Square Bottom flaskor, polypropen </td><td> Genesee Scientific </td><td> 32-130 </td><td></td></tr><tr><td> Flugs Förslutningar för Stock Flaskor </td><td> Genesee Scientific </td><td> 49-100 </td><td></td></tr><tr><td> DrosophilaFlaskor, Brett, polystyren </td><td> Genesee Scientific </td><td> 32-117 </td><td></td></tr><tr><td> Flugs Förslutningar för breda Injektionsflaskor </td><td> Genesee Scientific </td><td> 49-101 </td><td></td></tr><tr><td> Wide Mynningsstorlekar Aardvark pipettspetsar, 200 ul </td><td> Denville Scientific </td><td> P1105-CP </td><td></td></tr><tr><td> Flystuff Flypad, standardstorlek </td><td> Genesee Scientific </td><td> 59-114 </td><td></td></tr><tr><td> BD Falcon 15 ml koniska centrifugrör </td><td> Fisher Scientific </td><td> 14-959-70C </td><td></td></tr><tr><td> Fisherbrand petriskålar med tydliga Lock, 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 Kolvar 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> Öljäst </td><td> MP Biomedicals </td><td> 0290331280 </td><td></td></tr><tr><td> STRÖSOCKER </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 konserveringsmedel i livsmedel </td></tr><tr><td> Propionsyra, 99% </td><td> Acros Organics </td><td> 149300025 </td><td> Fly konserveringsmedel i livsmedel </td></tr><tr><td> Kanamycinsulfat </td><td> ISC BioExpress </td><td> 0408-10G </td><td></td></tr><tr><td> Tetracyklin HCl </td><td> VWR </td><td> 80058-724 </td><td></td></tr></tbody></table>

measurement of lifespan in drosophila melanogaster

Åldrande är ett fenomen som resulterar i jämn fysiologisk försämring nästan alla organismer som har undersökts, vilket leder till minskad fysisk prestationsförmåga och ökad risk för sjukdom. Individuell åldrande är uppenbar på populationsnivå som en ökning av åldersberoende dödlighet, som ofta mäts i laboratoriet genom att observera livslängd i stora kohorter av åldersmatchade individer. Experiment som syftar till att kvantifiera i vilken utsträckning genetisk eller miljömässig manipulationer påverkar livslängden i enkla modellorganismer har varit anmärkningsvärt framgångsrika för att förstå de aspekter av åldrandet som är konserverade mellan taxa och inspirerande nya strategier för att utöka livslängden och förhindra ålder-associerad sjukdom hos däggdjur . Ättika flyga, Drosophila melanogaster, är en attraktiv modell organism för att studera mekanismerna för åldrande på grund av dess relativt korta livslängd, bekväm djurhållning, och enkel genetik.Men demografiska mått på åldrande, inklusive åldersspecifik överlevnad och dödlighet, är utomordentligt känsliga för även små variationer i experimentell design och miljö, och upprätthållandet av strikta laboratoriepraxis så länge åldrande experiment krävs. Dessa överväganden tillsammans med behovet att öva noggrann kontroll av genetisk bakgrund, är avgörande för att generera robusta mätningar. Det finns faktiskt många anmärkningsvärda kontroverser kring slutledning från livslängd experiment i jäst, maskar, flugor och möss som har spårats till miljö-eller genetiska artefakter 1-4. I detta protokoll beskriver vi en rad förfaranden som har optimerats under många år för att mäta livslängd i Drosophila med laboratorium flaskor. Vi beskriver också användningen av dLife programvara som har utvecklats av vårt laboratorium och finns för nedladdning ( <a href="http://sitemaker.umich.edu/pletcherlab/software">http://sitemaker.umich.edu/ple</a>tcherlab / mjukvara). dLife accelererar genomströmningen och främjar god praxis genom att införliva optimal experimentell design, vilket förenklar flyga hantering och insamling av data, och standardisera dataanalys. Vi kommer också att diskutera de många potentiella fallgropar i utformningen, insamling och tolkning av livslängd data och vi tillhandahåller åtgärder för att undvika dessa faror.

Ett enklare system av protokollet presenteras i figur 1, där viktiga steg beskrivs. Synkroniseringen del av protokollet kan användas för olika analyser som kräver åldersmatchade vuxna flugor. Typiska survivorship kurvorna för vildtyp flugor visas i figur 2a, med användning av programvaran dLife experimentet hantering (figur 2b, c). Vuxna hanar lever oftast kortare, med båda populationerna uppnå en genomsnittlig och median livslängd...

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

Measurement of Lifespan in Drosophila melanogaster

Drosophila melanogaster är en kraftfull modell organism för att utforska den molekylära grunden för livslängd reglering. Detta protokoll kommer att diskutera de olika stegen i generering av en reproducerbar, populationsbaserade mätning av livslängden samt potentiella fallgropar och hur man undviker dem.

Mätning av livslängden 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 ...

measurement-of-lifespan-in-drosophila-melanogaster

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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 examine the expression of various novel and marker proteins.  Staining of whole larvae is impractical because the tough cuticle prevents antibodies from penetrating inside the body cavity.  In order to stain these tissues it is necessary to dissect the animal prior to fixing and staining.  In this article we demonstrate how to dissect Drosophila larvae without damaging the CNS.  Begin by tearing the larva in half with a pair of fine forceps, and then turn the cuticle "inside-out" to expose the CNS.  If the dissection is performed carefully the CNS will remain attached to the cuticle.  We usually keep the CNS attached to the cuticle throughout the fixation and staining steps, and only completely remove the CNS from the cuticle just prior to mounting the samples on glass slides.  We also show some representative images of a larval CNS stained with Eve, a transcription factor expressed in a subset of neurons in the CNS.  The article concludes with a discussion of some of the practical uses of this technique and the potential difficulties that may arise.

In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae.

Dissekering av Larvernas CNS i 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 locomotor behavior in Drosophila melanogaster relies on the ability to be able to quantify changes in motion during or in response to a given task. For this reason, a high-resolution video tracking system, such as the one we describe in this paper, is a valuable tool for measuring locomotion in real-time. Our protocol involves the use of an initial air pulse to break the flies momentum, followed by a thirty second filming period in a square chamber. A tracking program is then used to calculate the instantaneous speed of each fly within the chamber in 10 msec increments. Analysis software then compiles this data, and outputs a variety of parameters such as average speed, max speed, time spent in motion, acceleration, etc. This protocol will discuss proper feeding and management of flies for behavioral tasks, handling flies without anesthetization or immobilization, setting up a controlled environment, and running the assay from start to finish.

The study of complex locomotor behavior in Drosophila melanogaster is dependent upon the ability to quantify changes in a given fly's movement. This article demonstrates how to do this using a high-resolution tracking system.

Högupplöst video Spårning av rörelseorgan hos vuxna 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 ...

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 present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system1 we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo. Following collection of embryos at the desired stage of development, the outer chorion is removed and the embryos are then mounted in halocarbon oil between a hydrophobic, gas-permeable membrane and a glass coverslip for live imaging. In addition to gross migratory parameters such as speed and directionality, higher resolution imaging coupled with the use of fluorescent reporters of F-actin and microtubules can provide more detailed information concerning the dynamics of these cytoskeletal components.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

Live avbildning av Drosophila melanogaster Embryonala Hemocyte Flyttningar

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

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides protection against desiccation and other environmental challenges. Several of these cuticular hydrocarbon (CHC) compounds also function as semiochemical signals, and as such mediate pheromonal communications between members of the same species, or in some instances between different species, and influence behavior. Specialized cells referred to as oenocytes are regarded as the primary site for CHC synthesis. However, relatively little is known regarding the involvement of the oenocytes in the regulation of the biosynthetic, transport, and deposition pathways contributing to CHC output. Given the significant role that CHCs play in several aspects of insect biology, including chemical communication, desiccation resistance, and immunity, it is important to gain a greater understanding of the molecular and genetic regulation of CHC production within these specialized cells. The adult oenocytes of D. melanogaster are located within the abdominal integument, and are metamerically arrayed in ribbon-like clusters radiating along the inner cuticular surface of each abdominal segment. In this video article we demonstrate a dissection technique used for the preparation of oenocytes from adult D. melanogaster. Specifically, we provide a detailed step-by-step demonstration of (1) how to fillet prepare an adult Drosophila abdomen, (2) how to identify the oenocytes and discern them from other tissues, and (3) how to remove intact oenocyte clusters from the abdominal integument. A brief experimental illustration of how this preparation can be used to examine the expression of genes involved in hydrocarbon synthesis is included. The dissected preparation demonstrated herein will allow for the detailed molecular and genetic analysis of oenocyte function in the adult fruit fly.

Dissection of Oenocytes from Adult Drosophila melanogaster

In insects, the oenocytes produce cuticular hydrocarbon compounds. These compounds protect against desiccation and facilitate chemical communication. Here we demonstrate a dissection technique used to isolate the oenocytes from adult Drosophila melanogaster, and illustrate how this preparation can be utilized to study genes involved in hydrocarbon synthesis.

Dissekering av Oenocytes från Vuxen Drosophila melanogaster</em

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

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline interactions. Testes are typically isolated from adult males 0-3 days after eclosion from the pupal case. The testes of wild-type flies are easily distinguished from other tissues because they are yellow, but the testes of white mutant flies, a common genetic background for laboratory experiments are similar in both shape and color to the fly gut. Performing dissection on a glass microscope slide with a black background makes identifying the testes considerably easier. Testes are removed from the flies using dissecting needles. Compared to protocols that use forceps for testes dissection, our method is far quicker, allowing a well-practiced individual to dissect testes from 200-300 wild-type flies per hour, yielding 400-600 testes. Testes from white flies or from mutants that reduce testes size are harder to dissect and typically yield 200-400 testes per hour.

Isolation of Drosophila melanogaster Testes

Drosophila melanogaster testes can be rapidly and efficiently isolated from adult males using dissecting needles. With practice, one can readily isolate in one or two days an amount of testes sufficient for the analysis of DNA or RNA by high throughput sequencing or more traditional molecular biology methods or of protein for antibody- or enzyme-based assays.

Isolering av Drosophila melanogaster Testiklar

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

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled analysis of cell and developmental biology from a variety of methodological angles. Live imaging is an emerging method for observing dynamic cell processes, such as cell division or cell motility. Having isolated mutations in uncharacterized putative cell cycle proteins it became essential to observe mitosis in situ using live imaging. Most live imaging studies in Drosophila have focused on the embryonic stages that are accessible to manipulation and observation because of their small size and optical clarity. However, in these stages the cell cycle is unusual in that it lacks one or both of the gap phases. By contrast, cells of the pupal wing of Drosophila have a typical cell cycle and undergo a period of rapid mitosis spanning about 20 hr of pupal development. It is easy to identify and isolate pupae of the appropriate stage to catch mitosis in situ. Mounting intact pupae provided the best combination of tractability and durability during imaging, allowing experiments to run for several hours with minimal impact on cell and animal viability. The method allows observation of features as small as, or smaller than, fly chromosomes. Adjustment of microscope settings and the details of mounting, allowed extension of the preparation to visualize membrane dynamics of adjacent cells and fluorescently labeled proteins such as tubulin. This method works for all tested fluorescent proteins and can capture submicron scale features over a variety of time scales. While limited to the outer 20 &#181;m of the pupa with a conventional confocal microscope, this approach to observing protein and cellular dynamics in pupal tissues in vivo may be generally useful in the study of cell and developmental biology in these tissues.

Imaging Through the Pupal Case of Drosophila melanogaster

This paper demonstrates the use of a fast scanning confocal microscope to image cell behavior directly through the puparium. By leaving the pupal case intact, this method allows observation and measurement of dynamic cell processes at a stage of Drosophila development that is difficult to study directly.

Imaging Genom pupal Fall av<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

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.

Mätning av ämnesomsättning i<em&gt; Drosophila</em&gt; Med respiration

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

The last decades have witnessed the explosion of scientific interest around gene expression control mechanisms at the RNA level. This branch of molecular biology has been greatly fueled by the discovery of noncoding RNAs as major players in post-transcriptional regulation. Such a revolutionary perspective has been accompanied and triggered by the development of powerful technologies for profiling short RNAs expression, both at the high-throughput level (genome-wide identification) or as single-candidate analysis (steady state accumulation of specific species). Although several state-of-art strategies are currently available for dosing or visualizing such fleeing molecules, Northern Blot assay remains the eligible approach in molecular biology for immediate and accurate evaluation of RNA expression. It represents a first step toward the application of more sophisticated, costly technologies and, in many cases, remains a preferential method to easily gain insights into RNA biology. Here we overview an efficient protocol (Enhanced Northern Blot) for detecting weakly expressed microRNAs (or other small regulatory RNA species) from Drosophila melanogaster whole embryos, manually dissected larval/adult tissues or in vitro cultured cells. A very limited amount of RNA is required and the use of material from flow cytometry-isolated cells can be also envisaged.

Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster

The aim of this publication is to visualize and discuss the operative steps of an Enhanced Northern Blot protocol on RNA extracted from Drosophila melanogaster embryos, cells, and tissues. This protocol is particularly useful for the efficient detection of small RNA species.

Förbättrad Northern blot detektering av små RNA arter 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

Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life cycle of Drosophila enables collection of large quantities of material from embryos, larvae, pupae and adult flies, in a synchronized way, at a low economic cost. A major strategy for propagating large numbers of flies is the use of a fly population cage. This useful and common tool in the Drososphila community is an efficient way to regularly produce milligrams to tens of grams of embryos, depending on uniformity of developmental stage desired. While a population cage can be time consuming to set up, maintaining a cage over months takes much less time and enables rapid collection of biological material in a short period. This paper describes a detailed and flexible protocol for the maintenance of a Drosophila melanogaster population cage, starting with 1.5 g of harvested material from the previous cycle.

Maintenance of a Drosophila melanogaster Population Cage

This manuscript reports a detailed protocol for culturing, on a regular basis, a population of Drosophila melanogaster using a fly population cage.

Underhåll av 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 ...

Measurement of Carbon Dioxide Production from Radiolabeled Substrates in Drosophila melanogaster

The power of Drosophila genetics is increasingly being applied to questions of hormone signaling and metabolism and to the development of models of human disease in this organism. Sensitive methods for measurements of parameters such as metabolic rates are needed to drive the understanding of physiology and disease in small animals such as the fruit fly. The method described here assesses fuel oxidation in small numbers of adult flies fed food containing trace amounts of 14C-labeled substrates such as glucose or fatty acid. After the feeding period and any additional experimental manipulations, flies are transferred to short tubes capped with mesh, which are then placed in glass vials containing KOH-saturated filter paper that traps exhaled, radiolabeled CO2 generated from oxidation of radiolabeled substrates as potassium bicarbonate, KHCO3. This radiolabeled bicarbonate is measured by scintillation counting. This is a quantitative, reproducible, and simple approach for the study of fuel oxidation. The use of radiolabeled glucose, fatty acids, or amino acids allows determination of the contribution of these different fuel sources to energy metabolism under different conditions such as feeding and fasting and in different genetic backgrounds. This complements other approaches used to measure in vivo energy metabolism and should further the understanding of metabolic regulation.

Measurement of Carbon Dioxide Production from Radiolabeled Substrates in Drosophila melanogaster

This paper describes a method for the measurement of fuel oxidation in Drosophila melanogaster in which trace amounts of specific radiolabeled metabolic substrates are fed to flies. The exhaled radiolabeled CO2 that is a produced from fuel oxidation is collected and measured.

Mätning av koldioxid Produktionen från Radiomärkt substrat i<em&gt; Drosophila melanogaster</em

The power of Drosophila genetics is increasingly being applied to questions of hormone signaling and metabolism and to the development of models of human ...