Name: imaging through the pupal case of drosophila melanogaster
Uploaded: 2014-01-23T15:15:00
Description: Watch this Scientific Journal Video about Imaging Through the Pupal Case of Drosophila melanogaster at JoVE.com

Forfatterne ønsker at anerkende Akira Chiba for intellektuel støtte, materiel støtte, og lagre. Tak til Julia Dallman for kommentarer.

1.. Flyv Arbejde <ol><li> Vedligehold fluer på standard cornmeal-agar-melasse-gær medium ved stuetemperatur 10. </li><li> For kors, isolere jomfruer inden 6 timer af eclosion. Efter at have krydset til hanner af den ønskede genotype, fluer skift til nye hætteglas hver 3-4 dage. </li></ol> Bemærk: Til disse eksperimenter blev Gal4 linje A9 anvendes til at drive ekspression af transgener i vingen. Flyve lagre kan fås fra bestanden center i Bloomington. Bestande, der anvendes i di...

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At visualisere, måle og kvantificere funktioner i delende celler, der kræves udvikling af en enkel bearbejdning for at observere mitose i levende puppe fløj af Drosophila ved hjælp af konfokal analyse af His2AvGFP udtrykkende celler. Denne metode blev anvendt til at dokumentere, at cellecyklus i puppe fløj bærer stærke ligheder med cellecykler i pseudostratified epiteler i at kerner flytte til den apikale overflade af epitel hvor de kommer ind mitose. Efter telofase, kerner falde tilbage i epitellaget. D...

<html> Den eddike flyve, Drosophila melanogaster, er en veletableret model til at studere mange aspekter af biologi. Drosophila forskning har en rig historie af genetiske eksperimenter, der tillader sofistikerede former for genmanipulation, herunder ytringsfrihed, knockdown og mutation. Med fremkomsten af ​​fluorescerende mærker protein, har dette repertoire udvidet til at omfatte undersøgelser af celler og proteiner i levende dyr. Fluen embryo er et glimrende system til sådanne undersøgelser, som det er lille og optisk klar tillader dyb, høj opløsning billeddannelse in vivo 1-3. Andre stadier af flue udvikling har vist sig at være mindre medgørlig, der kræver bedøvelse 4, dissektion og kortsigtet kultur 5,6, eller oprettelsen af vinduer i neglebånd til billeddannelse 7,8. Disse manipulationer normalt kompromittere udviklingen dyr på lang sigt eller påvirke dyr på måder, der begrænser billedbehandling til korte perioder. ent &quot;&gt; At studere nye mutationer i gener, der ligner cellecyklus regulatorer, var det nødvendigt at finde en passende forberedelse til at studere timingen og troskab af cellecyklus. Da de fleste embryonale cellecykler bliver afkortet (SM eller S-G2-M) og mutanterne under undersøgelse ikke viser fejl i senere stadier, var det vigtigt at overholde cellecyklussen i puppestadiet væv. Epitelceller i puppe har en mere typisk G1-S-G2-M cellecyklus og pupper af denne fase er ikke kan muskelbevægelser 9. Den oprindelige udgangspunkt for manipulationer medtaget intakt hele pupper udtrykker Histone2AV-GFP. Trods den tilsyneladende opaciteten af puppe tilfælde dette intakt præparat vist sig at være fremragende til langvarig in vivo-billeddannelse. Denne teknik er enkel nok, at bachelor-forskere rutinemæssigt bruger det til at studere aspekter af celle-og udviklingsmæssige biologi i Drosophila og alligevel opløsningen er fint nok til at tillade diskrimination af mikrometer skala funktioner. Med denne metode er mulige observationer af begivenheder over timer, minutter eller sekunder blot ved at justere tidsrække parametre. Videoer ved hjælp af blå, grøn, gul, orange og røde fluorescerende proteiner, eller kombinationer af disse, er blevet foretaget. Vigtigere er, hvis draget omsorg for at minimere laser intensitet, selv langsigtede billeddannelse har ingen effekt på udvikling eller levedygtighed af dyrene.

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		<col />
		<col />
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	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Fly stuff fly pad</td>
			<td style="width:71px;">Genesee scientific</td>
			<td style="width:119px;">59-114</td>
			<td style="width:167px;">for fly anesthetization</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">CO2 gas</td>
			<td style="width:71px;">Airgas south</td>
			<td style="width:119px;">CD50</td>
			<td>For fly anesthetization</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Regulator</td>
			<td style="width:71px;">Airgas south</td>
			<td style="width:119px;"> </td>
			<td>CO2 regulator</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Fly vials</td>
			<td style="width:71px;">Genesee scientific</td>
			<td>32-113RLBF</td>
			<td>Fly culture</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">Drosophila lines:A9-Gal4 (Bl#8761), His2Av-GFP (Bl#5941), Sco/CyO HsCre (Bl#1092), UAS-ChRFP-Tub (Bl#25773)</td>
			<td style="width:71px;">Bloomington Stock center</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Glass bottom dishes #1 1/2</td>
			<td style="width:71px;">WillCo Wells BV</td>
			<td style="width:119px;"> </td>
			<td>For microscopy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Thiodiethylene Glycol</td>
			<td style="width:71px;">Fluka</td>
			<td align="right" style="width:119px;">88559</td>
			<td>mountant</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Modeling clay</td>
			<td style="width:71px;">art supply store</td>
			<td style="width:119px;"> </td>
			<td>Support to position pupae against</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Paintbrushes</td>
			<td style="width:71px;">art supply store</td>
			<td style="width:119px;"> </td>
			<td>To manipulate flies</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Fine Forceps, Inox #5</td>
			<td style="width:71px;">Fine science tools</td>
			<td style="width:119px;">11252-20</td>
			<td>Dumont #5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">computer</td>
			<td style="width:71px;">any</td>
			<td style="width:119px;"> </td>
			<td>8 Gb RAM for image/movie analysis</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:47px;width:216px;">Fiji software</td>
			<td style="width:71px;"> </td>
			<td style="width:119px;">Free ware http://fiji.sc/Fiji</td>
			<td>Image analysis software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Confocal microscope</td>
			<td style="width:71px;"> </td>
			<td style="width:119px;"> </td>
			<td>Any fast scanning confocal should be sufficient</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">20x dry, and 40x or 63x oil immersion lenses</td>
			<td style="width:71px;">any</td>
			<td style="width:119px;"> </td>
			<td>For imaging tissue, cellular and subcellular features</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immersion oil (non fluorescent)</td>
			<td style="width:71px;"> </td>
			<td style="width:119px;"> </td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Stereo microscope</td>
			<td style="width:71px;">any</td>
			<td style="width:119px;"> </td>
			<td>For fly manipulation</td>
		</tr>
	</tbody>
</table>

imaging through the pupal case of drosophila melanogaster

Den mangeårige brug af Drosophila som model for celle-og udviklingsmæssige biologi har givet en bred vifte af værktøjer. Sammen har disse teknikker aktiveret analyse af celle-og udviklingsmæssige biologi fra en række forskellige metodiske vinkler. Live-billeddannelse er en ny metode til at observere dynamiske celle processer såsom celledeling eller celle motilitet. Efter at have isoleret mutationer i uncharacterized formodede cellecyklusproteiner det blev vigtigt at overholde mitose in situ ved hjælp af direkte billedvisning. De fleste levende billeddiagnostiske undersøgelser i Drosophila har fokuseret på fosterstadiet, der er tilgængelige for manipulation og observation på grund af deres lille størrelse og optisk klarhed. Men i disse faser af cellecyklus er usædvanligt ved, at det mangler en af ​​de gap faser eller begge dele. Derimod celler af puppe fløj af Drosophila har en typisk cellecyklus og gennemgå en periode med hurtig mitose spænder 20 timer af puppe udvikling. Det er let at jegdentify og isolere pupper af den pågældende fase at fange mitose in situ. Montering intakt pupper forudsat den bedste kombination af medgørlighed og holdbarhed under billedbehandling, så eksperimenter for at køre i flere timer med minimal indvirkning på celle-og dyre-levedygtighed. Fremgangsmåden tillader observation af funktioner så lille som, eller mindre end, flyve kromosomer. Justering af mikroskop indstillinger og detaljerne i montage, tillod udvidelse af præparatet til at visualisere membran dynamik tilstødende celler og fluorescens-mærkede proteiner såsom tubulin. Denne metode virker for alle testede fluorescerende proteiner og kan fange submicron skala funktioner over en bred vifte af tidsskalaer. Mens begrænset til den ydre 20 um puppe med et konventionelt konfokalt mikroskop, kan denne tilgang til at observere protein og cellulære dynamik puppe væv in vivo generelt være nyttige i studiet af cellen og udviklingsmæssige biologi i disse væv.

Celler i pseudostratified epitel, såsom udvikling af Drosophila øjet eller den ventrikulære lag i udviklingslandene hvirveldyr centralnervesystemet undergår nukleare bevægelser, betegnet interkinetic nuklear migration, i takt med cellecyklus. DNA-replikation forekommer, når kerner er ved eller nær den basale overflade og celler ind mitose, når kernerne nå apikale overflade 15,16. De puppe fløj celler danner et hurtigt delende monolag epitel i de første mange timer efter hoved udkrængning....

Watch this Scientific Journal Video about Imaging Through the Pupal Case of Drosophila melanogaster at JoVE.com

Imaging Through the Pupal Case of Drosophila melanogaster

Dette dokument viser anvendelsen af ​​en hurtig konfokalt mikroskop billedet celle adfærd direkte gennem puparium. Ved at lade puppe tilfælde intakt, giver denne metode observation og måling af dynamiske celleprocesser i en fase af Drosophila udvikling, der er vanskeligt at studere direkte.

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 ...

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Biology

Imaging Through the Pupal Case of 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 ...

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 ...

Upright Imaging of Drosophila Embryos

Upright Imaging of Drosophila Embryos

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current ...

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 ...

Drosophila Pupal Abdomen Immunohistochemistry

Drosophila Pupal Abdomen Immunohistochemistry

The Drosophila pupal abdomen is an established model system for the study of epithelial morphogenesis and the development of sexually dimorphic ...

Measurement of Lifespan in Drosophila melanogaster

Measurement of Lifespan in Drosophila melanogaster

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 ...

Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

Ex vivo kultur Drosophila puppe Testikel og Single Mand Kim-line cyster: Dissektion, Imaging, og farmakologisk behandling

During spermatogenesis in mammals and in Drosophila melanogaster, male germ cells develop in a series of essential developmental processes. This includes ...

Dissection and Mounting of Drosophila Pupal Eye Discs

Dissection and Mounting of Drosophila Pupal Eye Discs

Dissektion og Montering af<em&gt; Drosophila</em&gt; puppe Eye Discs

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes.  It contains approximately 800 ...

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 ...