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

この作品は、エリソン医学財団（SDP、からの資金によって支えられ<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）とNIH R01AG030593（SDP）。この作品は、P30-AG-013283（老化の国立研究所によって資金を供給され、老化の生物学における卓越性のネイサンショックセンターのショウジョウバエ老化コア（DAC）のリソースを活用し<a href="http://www.nih.gov" target="_blank">http://www.nih.gov/を</a> ）。著者は、原稿の批判的な読みのために特に有用な議論とブライアンチョンのためPletcher研究所に感謝したいと思います。我々は、データ収集の支援についてはニックAsherとキャスリンBorowiczを承認したいと思います。

我々は、4℃でプロトコルに現れる実験食品、酵母ペースト、ぶどう寒天プレートを格納し、カビや乾燥が幼虫と成虫の両方の標準環境条件をインチに設定していない限り、1〜2ヵ月以内にそれらを使用することをお勧めします12時12時間の明暗サイクルおよび60％の相対湿度で25℃のインキュベーター中でハエのメンテナンスを伴うステージングします。 1。実験食の調製</p...

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ここで紹介するプロトコルは、遺伝薬理学的、および環境的介入の評価のために適応可能であるショウジョウバエの成虫寿命の再現性のある測定値を生成するための方法を説明します。プロトコルの重要な側面は、慎重に、幼虫の開発環境を制御する大人のストレスを最小限にし、実験群とコントロールの間にバイアスを最小限に含まれています。またdLife寿命実験管理ソフトウェア?...

<table border="1"><tbody><tr><td> 試薬の名称 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント（オプション） </td></tr><tr><td>ドライイースト</td><td> Fleishmann酵母</td><td> 2192 </td><td></td></tr><tr><td>グレープ寒天粉末プレミックス</td><td>ジェネシー科学</td><td> 47から102 </td><td></td></tr><tr><td>大胚コレクションケージ</td><td>ジェネシー科学</td><td> 59から101 </td><td></td></tr><tr><td>大型交換エンドキャップ</td><td>ジェネシー科学</td><td> 59から103 </td><td></td></tr><tr><td> 6オンススクエアボトムボトル、ポリプロピレン</td><td>ジェネシー科学</td><td> 32から130 </td><td></td></tr><tr><td>ストックボトル用Flugsクロージャ</td><td>ジェネシー科学</td><td> 49から100 </td><td></td></tr><tr><td> ショウジョウバエバイアル、ワイド、Polystrene </td><td>ジェネシー科学</td><td> 32から117 </td><td></td></tr><tr><td>ワイドバイアル用Flugsクロージャ</td><td>ジェネシー科学</td><td> 49から101 </td><td></td></tr><tr><td>ワイドオリフィスアリクイのピペットチップ、200μlの</td><td>デンヴィル科学</td><td> P1105-CP </td><td></td></tr><tr><td> Flystuff Flypad、標準サイズ</td><td>ジェネシー科学</td><td> 59から114 </td><td></td></tr><tr><td> BDファルコン15mlコニカル遠心管</td><td>フィッシャー·サイエンティフィック</td><td> 14から959-70C </td><td></td></tr><tr><td>クリア蓋、隆起とFisherbrandペトリ皿、100径×15 mm（高さ）; </td><td>フィッシャー·サイエンティフィック</td><td> 08-757-12 </td><td></td></tr><tr><td> Kimax * Colorwareフラスコ千ミリリットル黄色</td><td>フィッシャー·サイエンティフィック</td><td> 10-200-47 </td><td></td></tr><tr><td> PBSのpHは7.4〜10倍</td><td>でvitrogen </td><td> 70011044 </td><td></td></tr><tr><td>テングサ寒天</td><td> Mooragar </td><td> N / A </td><td></td></tr><tr><td>ビール酵母</td><td> MPバイオメディカル</td><td> 0290331280 </td><td></td></tr><tr><td>グラニュー糖</td><td>クローガー</td><td> N / A </td><td></td></tr><tr><td> Tegosept </td><td>ジェネシー科学</td><td> 20から266 </td><td>食品防腐剤を飛ばす</td></tr><tr><td>プロピオン酸、99％ </td><td>アクロスオーガニック</td><td> 149300025 </td><td>食品防腐剤を飛ばす</td></tr><tr><td>硫酸カナマイシン</td><td> ISC BioExpress </td><td> 0408-​​10G </td><td></td></tr><tr><td>テトラサイクリン塩酸</td><td> VWR </td><td> 80058-724 </td><td></td></tr></tbody></table>

measurement of lifespan in drosophila melanogaster

高齢化は、それが検討されたほぼすべての生物が順調に生理的な劣化の結果は、減少した身体能力と疾患のリスクの増加につながるという現象である。個々の高齢化は、多くの場合、年齢をマッチさせた個体の大規模コホートで寿命を観察することにより、実験室で測定され、年齢に依存死亡率の増加は、人口レベルでのマニフェストです。単純なモデル生物でどの遺伝や環境操作衝撃寿命の程度を定量化しようとする実験が寿命を延長し、哺乳類では加齢に伴う疾患を予防するための分類群を横切って、感激新しい戦略のために保存されており、高齢化の側面を理解するために著しく成功してきた。 ショウジョウバエ、 キイロショウジョウバエは 、その比較的短い寿命、便利業、そして容易な遺伝に起因する老化のメカニズムを研究するための魅力的なモデル生物である。しかし、年齢別生存率と死亡率を含め、高齢化の人口学的指標は、実験的なデザインと環境のわずかな変化に非常に敏感である、老化実験期間中の厳格な検査室の安全基準の整備が必要となります。これらの考慮事項は、一緒に遺伝的背景を慎重にコントロールを練習する必要性と、堅牢な測定値を生成するために不可欠です。確かに、環境や遺伝的アーティファクト1-4にトレースされている酵母、ワーム、ハエやマウスの長寿実験から推論を取り巻く多くの著名な論争があります。このプロトコルでは、我々は、実験室のバイアルを使用して、ショウジョウバエの寿命を測定することが長年にわたって最適化されている一連の手順を説明します。私達はまた私達の研究室で開発され、ダウンロード（利用可能ですdLifeたソフトウェアの使用を記述<a href="http://sitemaker.umich.edu/pletcherlab/software">http://sitemaker.umich.edu/ple</a>tcherlab /ソフトウェア）。 dLifeは、スループットを加速し、フライ処理とデータ収集の簡素化、データ解析の標準化、最適な実験デザインを組み込むことにより、優れた実践を推進しています。また、設計、収集、および寿命のデータの解釈には多くの潜在的な落とし穴について説明します、我々はこれらの危険性を回避するための手順を提供します。

プロトコルの簡略化スキームが重要なステップが概説されて、 図1に示されている。プロトコルの同期部分は、年齢をマッチさせた成虫の​​ハエを必要とする様々なアッセイのために使用することができます。 野生型ハエの典型的な生存曲線はdLife実験管理ソフトウェア（ 図2b、c）を用いて、 図2aに示されています。成人男性は通...

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

Measurement of Lifespan in Drosophila melanogaster

キイロショウジョウバエは寿命調節の分子基盤を探索するための強力なモデル生物である。このプロトコルは、長寿の再現性は、人口ベースの測定だけでなく、潜在的な落とし穴とその回避方法を生成するために必要な手順を説明します。

における寿命の測定<em&gt;キイロショウジョウバエ</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

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 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.

キイロショウジョウバエの幼虫中枢神経系の解剖

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

生物学

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.

アダルトキイロショウジョウバエのロコモーションの高解像度のビデオトラッキング

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.

のライブイメージングキイロショウジョウバエ胚性血球移行

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.

大人からOenocytesの解剖キイロショウジョウバエ</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.

の分離キイロショウジョウバエ精巣

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.

の蛹ケースを画像化<em&gt;キイロショウジョウバエ</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.

代謝率の測定に<em&gt;ショウジョウバエ</em&gt;呼吸計測を使用した

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.

低分子RNA種のノーザンブロット検出における強化<em&gt;キイロショウジョウバエ</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.

のメンテナンス<em&gt;キイロショウジョウバエ</em&gt;人口ケージ

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.

放射性標識基質からの二酸化炭素生成での測定<em&gt;キイロショウジョウバエ</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 ...