Name: a buoyancy-based method of determining fat levels in drosophila
Uploaded: 2016-11-01T11:15:00
Description: Watch this Scientific Journal Video about A Buoyancy-based Method of Determining Fat Levels in Drosophila at JoVE.com

K.E.H. was supported by the Training Grant in Molecular Biology NIH-T32-GM08730. This work was supported by NIH, NIDDK Grant 5K01DK095932 and AHA Award 12BGIA11930014 to T.R.

1. Samle egg fra Flies med genotyper av interesse MERK: Ideell kors består av 150 jomfru fluer og minst 75 hanner. Aksje samlinger bør bestå av minst 200 fluer. <ol><li> Forbered drue plater. <ol><li> Legg 37,5 g agar til 1,5 liter destillert vann i en 4 liters kolbe. </li><li> Autoklav vann / agar blanding i 50 min ved 121 ºC (250 ºF). </li><li> Mens vann / agar mix er autoklave, tilsett 50 g sukrose til 500 ml druesaft og rør med en rørepinne på et oppvarmet rør plate før sukrose er oppløst. </li>...

<ol>
	<li>Bier, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila,+the+golden+bug,+emerges+as+a+tool+for+human+genetics.">Drosophila, the golden bug, emerges as a tool for human genetics.</a> Nat Rev Genet. 6, 9-23 (2005).</li><li>Liu, Z., Huang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+metabolism+in+Drosophila+development+and+disease+Using+Drosophila+System+to+Study+Lipid+Metabolism+Lipids+Function+in+Drosophila+Early+Development.">Lipid metabolism in Drosophila development and disease Using Drosophila System to Study Lipid Metabolism Lipids Function in Drosophila Early Development.</a> Acta Biochim Biophys Sin. 45 (1), 44-50 (2013).</li><li>Ruden, D. M., Lu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+conservation+of+metabolism+explains+howDrosophila+nutrigenomics+can+help+us+understand+human+nutrigenomics.">Evolutionary conservation of metabolism explains howDrosophila nutrigenomics can help us understand human nutrigenomics.</a> Genes Nutr. 1 (2), 75-84 (2006).</li><li>Wolf, M. J., Rockman, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila,+genetic+screens,+and+cardiac+function.">Drosophila, genetic screens, and cardiac function.</a> Circ. Res. 109, 794-806 (2011).</li><li>Trinh, I., Boulianne, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+obesity+and+its+associated+disorders+in+Drosophila.">Modeling obesity and its associated disorders in Drosophila.</a> Physiology (Bethesda). 28 (2), 117-124 (2013).</li><li>Azeez, O. I., Meintjes, R., Chamunorwa, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fat+body,+fat+pad+and+adipose+tissues+in+invertebrates+and+vertebrates:+the+nexus.">Fat body, fat pad and adipose tissues in invertebrates and vertebrates: the nexus.</a> Lipids Health Dis. 13, 71 (2014).</li><li>Dourlen, P., Sujkowski, A., Wessells, R., Mollereau, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fatty+acid+transport+proteins+in+disease:+New+insights+from+invertebrate+models.">Fatty acid transport proteins in disease: New insights from invertebrate models.</a> Prog. Lipid Res. 60, 30-40 (2015).</li><li>Hildebrandt, A., Bickmeyer, I., K&#252;hnlein, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reliable+Drosophila+body+fat+quantification+by+a+coupled+colorimetric+assay.">Reliable Drosophila body fat quantification by a coupled colorimetric assay.</a> PLoS One. 6 (9), e23796 (2011).</li><li>Tennessen, J. M., Barry, W. E., Cox, J., Thummel, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+studying+metabolism+in+Drosophila.">Methods for studying metabolism in Drosophila.</a> Methods. 68, 105-115 (2014).</li><li>Al-Anzi, B., Zinn, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colorimetric+measurement+of+triglycerides+cannot+provide+an+accurate+measure+of+stored+fat+content+in+Drosophila.">Colorimetric measurement of triglycerides cannot provide an accurate measure of stored fat content in Drosophila.</a> PLoS One. 5 (8), e12353 (2010).</li><li>Thanh, N. T. K., Stevenson, G., Obatoml, D., Bach, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+Lipids+in+Animal+Tissues+by+High-Performance+Thin-Layer+Chromatography+with+Densitometry.">Determination of Lipids in Animal Tissues by High-Performance Thin-Layer Chromatography with Densitometry.</a> J. Planar Chromatogr. 13, 375-381 (2000).</li><li>Borrull, A., Lopez-Martinez, G., Poblet, M., Cordero-Otero, R., Rozes, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+the+separation+and+quantification+of+neutral+lipid+species+using+GC-MS.">A simple method for the separation and quantification of neutral lipid species using GC-MS.</a> Eur. J. Lipid Sci. Technol. 117, 274-280 (2015).</li><li>Shui, G., 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=Toward+one+step+analysis+of+cellular+lipidomes+using+liquid+chromatography+coupled+with+mass+spectrometry:+application+to+Saccharomyces+cerevisiae+and+Schizosaccharomyces+pombe+lipidomics.">Toward one step analysis of cellular lipidomes using liquid chromatography coupled with mass spectrometry: application to Saccharomyces cerevisiae and Schizosaccharomyces pombe lipidomics.</a> Mol. Biosyst. 6, 1008-1017 (2010).</li><li>Farvid, M. S., Ng, T. W. K., Chan, D. C., Barrett, P. H. R., Watts, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+adiponectin+and+resistin+with+adipose+tissue+compartments,+insulin+resistance+and+dyslipidaemia.">Association of adiponectin and resistin with adipose tissue compartments, insulin resistance and dyslipidaemia.</a> Diabetes. Obes. Metab. 7 (4), 406-413 (2005).</li><li>Urbanchek, M. G., Picken, E. B., Kalliainen, L. K., Kuzon, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+force+deficit+in+skeletal+muscles+of+old+rats+is+partially+explained+by+the+existence+of+denervated+muscle+fibers.">Specific force deficit in skeletal muscles of old rats is partially explained by the existence of denervated muscle fibers.</a> J. Gerontol. A. Biol. Sci. Med. Sci. 56A (5), B191-B197 (2001).</li><li>Mosher, J., 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=Coordination+between+Drosophila+Arc1+and+a+specific+population+of+brain+neurons+regulates+organismal+fat.">Coordination between Drosophila Arc1 and a specific population of brain neurons regulates organismal fat.</a> Dev. Biol. 405 (2), 280-290 (2015).</li><li>Reis, T., van Gilst, M. R., Hariharan, I. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+buoyancy-based+screen+of+drosophila+larvae+for+fat-+storage+mutants+reveals+a+role+for+Sir2+in+coupling+fat+Storage+to+Nutrient+Availability.">A buoyancy-based screen of drosophila larvae for fat- storage mutants reveals a role for Sir2 in coupling fat Storage to Nutrient Availability.</a> PLoS Genet. 6 (11), e1001206 (2010).</li><li>Teixeira, L., Rabouille, C., R&#248;rth, P., Ephrussi, A., Vanzo, N. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+Perilipin/ADRP+homologue+Lsd2+regulates+lipid+metabolism.">Drosophila Perilipin/ADRP homologue Lsd2 regulates lipid metabolism.</a> Mech. Dev. 120 (9), 1071-1081 (2003).</li><li>Perez, C. L., Van Gilst, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+13C+Isotope+Labeling+Strategy+Reveals+the+Influence+of+Insulin+Signaling+on+Lipogenesis+in+C.+elegans.">A 13C Isotope Labeling Strategy Reveals the Influence of Insulin Signaling on Lipogenesis in C. elegans.</a> Cell Metab. 8, 266-274 (2008).</li></ol>

Det finnes et mangfold av teknikker som er utviklet for å måle lipid nivåer 8-10. Men hver av disse metodene kommer med noen store ulemper som er adressert av oppdrifts-baserte analysen beskrevet ovenfor. For det første er denne analysen ekstremt rask. Teste en full konsentrasjon gradient tar ikke mer enn 30 - 60 min. Dette er en stor forbedring på de fleste av de teknikker som er i bruk. For eksempel, lipid kvantitering ved MS tar 7 - 9 timer for å isolere lipider og ytterligere f...

Drosophila melanogaster har blitt brukt i over et århundre i studiet av genetikk og andre grunnleggende biologiske spørsmål. I de siste tiårene har det blitt klart at Drosophila er et kraftig verktøy i studiet av mange menneskelige sykdommer. Som 70 - 80% av gener assosiert med humane sykdommer har en identifisert fly ortolog til 1 - 4, gir Drosophila en forenklet, men translater system for å studere komplekse sykdommer. Metabolisme spesielt har dratt nytte av en slik studie. Ikke bare er genetikk av stoffskiftet godt konservert mellom fluer og mennesker, men de aktuelle organer og celletyper er også svært lik 2,5. For eksempel, fettet kroppen av flue butikkene begge triacylglycerides (TAG) og glykogen, funksjoner som er analoge med de som er utført i pattedyrs lever og hvite fettvev 6. Ved hjelp av Drosophila som en modell for menneskelig fedme er vesentlig forbedret vår forståelse av lipidmetabolisme og genetikk av fedme 7. Larveutviklingstrinn er spesielt nyttig for å studere segregering av næringsstoffer til lagring eller anvendelse som den er dedikert til mating og lagring av energi som skal brukes under pupariation. For tiden er det mange forskjellige kvantitative metoder for å bestemme fettlagringsnivåer i Drosophila. Den mest brukte metoden er koplet kolorimetrisk analyse (CCA) 8,9. CCA ble utviklet for å bestemme Tags nivåer i humant serum og opererer på den forutsetning at glyserol frigjort fra ryggraden av triglyserider vil gjennomgå en rekke reaksjoner, til slutt resulterer i en redox-reaksjon koblet generering av et farget produkt. Absorbansen av bestemte bølgelengder av lys måles så for å bestemme den initiale mengden av glycerol tilstede. Imidlertid kan glyserol også bli frigjort fra mono- og diacylglycerides i tillegg til TAG og kan derfor ikke være et nøyaktig mål på stored kroppsfett 9. Videre kan øye pigment av knust voksne fluer forstyrre enkelte avlesninger og komplisere resultater 9,10. Derfor CCA må ledsages og validert ved tynnsjiktskromatografi (TLC), som tillater separasjon av de fleste familier lipid som kan bli kvantifisert ved densitometri 10,11. Men kan noen lipidklasser som steroler ikke bli analysert og må tallfestes en annen måte 12. Massespektrometri (MS) er en nøyaktig måte å kvantifisere alle klasser av store cellulære lipider 12,13. Imidlertid lipid ekstraksjon prosedyrer som er nødvendige for å analysere med MS er både tidkrevende (mest tar nesten en hel dag) og kostbare. Her presenterer vi en alternativ metode for å raskt og billig bestemme organismefettinnhold i L3 larver av bananflue. Fremgangsmåten som presenteres nedenfor utnytter forskjellen i densitet mellom fettvev og magert vev. pattedyr fat vev har en tetthet på ca. 0,9 g / ml, 14 mens skjelettmuskulatur som en densitet på 1,06 g / ml 15. Denne forskjellen gjør at dyr med høyere lagre av fett vil ha lavere densitet enn dyr med lavere lagre av fett, som vil tillate dem å flyte bedre i en oppløsning av fast tetthet. Denne egenskapen gir svært rask screening av et stort antall dyr som samtidig er både billig og ikke-invasiv. Oppdrift basert analyse har blitt brukt både for å bekrefte fenotyper av å endre kjente regulatorer av fettnivåer samt å identifisere nye genetiske og nevrologiske regulatorer av fedme 16,17.

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			<td height="21" style="height:21px;width:216px;">Bacto Agar</td>
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			<td height="21" style="height:21px;width:216px;">Sucrose</td>
			<td style="width:168px;">Fisher</td>
			<td style="width:119px;">S512</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tegosept</td>
			<td style="width:168px;">Genesee Scientific</td>
			<td style="width:119px;">20-259</td>
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			<td height="21" style="height:21px;width:216px;">Ethanol</td>
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			<td>200 proof</td>
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			<td height="21" style="height:21px;width:216px;">Baker&#39;s yeast</td>
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			<td>Enriched degerminated</td>
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			<td height="21" style="height:21px;width:216px;">Drosophila Agar Type II</td>
			<td style="width:168px;">Apex</td>
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			<td height="21" style="height:21px;width:216px;">Propionic Acid</td>
			<td>VWR</td>
			<td>U330-09</td>
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			<td height="21" style="height:21px;width:216px;">Sodium chloride</td>
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			<td height="21" style="height:21px;">Potassium phosphate monobasic</td>
			<td>Sigma</td>
			<td>P5655-100G</td>
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			<td height="21" style="height:21px;width:216px;">Sodium phosphate anhydrous</td>
			<td style="width:168px;">Fisher</td>
			<td style="width:119px;">S3933</td>
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			<td height="21" style="height:21px;">Potassium chloride</td>
			<td>Fisher</td>
			<td>P330-500</td>
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			<td height="21" style="height:21px;width:216px;">35 x 10mm petri plate</td>
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			<td>08-757-100A</td>
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a buoyancy-based method of determining fat levels in drosophila

Drosophila melanogaster is a key experimental system in the study of fat regulation. Numerous techniques currently exist to measure levels of stored fat in Drosophila, but most are expensive and/or laborious and have clear limitations. Here, we present a method to quickly and cheaply determine organismal fat levels in L3 Drosophila larvae. The technique relies on the differences in density between fat and lean tissues and allows for rapid detection of fat and lean phenotypes. We have verified the accuracy of this method by comparison to body fat percentage as determined by neutral lipid extraction and gas chromatography coupled with mass spectrometry (GCMS). We furthermore outline detailed protocols for the collection and synchronization of larvae as well as relevant experimental recipes. The technique presented below overcomes the major shortcomings in the most widely used lipid quantitation methods and provides a powerful way to quickly and sensitively screen L3 larvae for fat regulation phenotypes while maintaining the integrity of the larvae. This assay has wide applications for the study of metabolism and fat regulation using Drosophila.

Figur 1 representerer et eksempel på hvordan den oppdrift-baserte analysen kan detektere både høyere og lavere fettlagre i genetisk manipulert larver. Figur 1A viser at eksitasjon eller stanse av en bestemt undergruppe av nevroner (E347) i larve hjernen induserer lavere og høyere fett butikker henholdsvis. Den samme genetisk bakgrunn kontroll ble benyttet i begge tilfeller. Figur 1B viser et eksempel på hvordan denne fenotype erhold...

Watch this Scientific Journal Video about A Buoyancy-based Method of Determining Fat Levels in Drosophila at JoVE.com

A Buoyancy-based Method of Determining Fat Levels in Drosophila

Her presenterer vi en metode for å måle organismefettnivåer i tredje stadium (L3) larvestadiet av bananflue. Denne fremgangsmåte utnytter den forholdsvis lave tetthet av fettvev for å skille mellom larver med endrede fettlagre. Oppdrift basert analyse er et verdifullt verktøy for rask, reproduserbar og økonomisk screening.

En Oppdrift-basert metode for å bestemme fettnivåer i<em&gt; Drosophila</em

Drosophila melanogaster is a key experimental system in the study of fat regulation. Numerous techniques currently exist to measure levels of stored fat ...

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A Buoyancy-based Method of Determining Fat Levels in Drosophila

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