Name: use of time lapse microscopy to visualize anoxia-induced suspended animation in c. elegans embryos
Uploaded: 2012-12-03T13:00:00
Description: Watch this Scientific Journal Video about Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos at JoVE.com

Vi vil gerne udtrykke vores påskønnelse for input og kommentarer fra medlemmer af Padilla Lab. Nematode stammer i dette arbejde blev leveret af Caenorhabditis Genetics Center, som er finansieret af NIH National Center for Forskning Ressourcer (NCRR). Vi anerkender og takker Dr. Lon Turnbull om teknisk bistand med konfokal mikroskopi. Dette arbejde er blevet støttet af en bevilling fra National Science Foundation (NSF-IOS, karriere) til PAP

1. Prøvefremstilling <ol><li> Producere eller indhente passende transgene C. elegans stamme af interesse ved hjælp af transgene metoder eller fra Caenorhabditis Genetics Stock Center (CGC) eller kollega. I dette tilfælde bruger vi stamme TH32 (pie-1 :: TBG-1 :: GFP, pie-1 :: GFP :: H2B) 5 til visualisere kromosomer og centrosomes som markører for celledeling. </li><li> Generer en synkroniseret befolkning ved hjælp af en af tre metoder: 1) smuldre drægtige voksne i hypochlori...

<ol>
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Ilt afsavn og suspenderet animation Skønt udsættelse for svær iltmangel kan være dødelig for visse organismer, nogle organismer er i stand til at overleve eksponering for iltmangel. I tilfælde af C. elegans, overlevelse af anoxi eksponering afhænger udviklingsstadiet og respons på iltmangel er trådt ind i en reversibel tilstand af suspenderet animation i hvilken en række observerbare biologiske processer bliver arresteret. Celledeling, udvikling, bevægelse, spise og reproduk...

<table border="1"><tbody><tr><td> Reagenser / Udstyr </td><td></td><td></td><td> Sammensætning </td></tr><tr><td> Hypochloritopløsning </td><td></td><td></td><td> 0,7 g KOH, 12 ml 5% NaOCl, bringe til 50 ml med ddH 2 0 </td></tr><tr><td> M9 buffer </td><td></td><td></td><td> 3 g KH 2 PO 4, 11 g Na 2 HPO 4, 5 g NaCI, 1 ml (1 M) MgSO4 per 1 L ddH 2 O </td></tr><tr><td> Mikroskopobjektglas </td><td> Fisher Scientific </td><td> 12-550-343 </td><td> 3 &quot;x1&quot; x1.0 mm </td></tr><tr><td> Round micro dækglas </td><td> Elektronmikroskopi Sciences </td><td> 72.223-01 </td><td> 25 mm Diameter </td></tr><tr><td> Halocarbon oil 700 </td><td> Sigma </td><td> H8898-100ml </td><td></td></tr><tr><td> Bedøvelsesmiddel </td><td></td><td></td><td> 0,5% triskAine, 0,05% tetramisol </td></tr><tr><td> Leiden Lukket Perfusion Microincubator </td><td> Harvard Apparatus </td><td> 650.041 </td><td></td></tr><tr><td> UHP Nitrogen </td><td> Calgaz (Air Liquide) </td><td></td><td> &gt; 99,9990% N2 &lt;2 ppm O2 </td></tr><tr><td> Plastrør </td><td> VWR </td><td> 89068-468 </td><td> 0,062 &quot;ID x 0,125&quot; OD </td></tr><tr><td> Roterende skive konfokalt mikroskop </td><td> McBain Systems </td><td></td><td> Zeiss inverteret optisk mikroskop, epifluorescens belysningssystem, CSU-10 Yokogawa konfokal skanner, Hamamatsu elektronmultiplikator CCD-kamera. </td></tr></tbody></table>

use of time lapse microscopy to visualize anoxia-induced suspended animation in c. elegans embryos

Caenorhabdits elegans er blevet udbredt i studiet af stress modstand, som lettes ved gennemskueligheden af voksne og embryo faser samt af tilgængeligheden af genetiske mutanter og transgene stammer, der udtrykker et utal af fusionsproteiner 1-4. Desuden kan dynamiske processer såsom celledeling ses med fluorescensmærkede reportermolekyler proteiner. Studiet af mitose kan lettes ved brug af time-lapse eksperimenter i forskellige systemer, herunder intakte organismer, og dermed den tidlige C. elegans embryo er velegnet til denne undersøgelse. Præsenteres her er en teknik, som in vivo-afbildning af sub-cellulære strukturer som reaktion på anoxiske (99,999% N2, &lt;2 ppm O2) stress er muligt ved hjælp af en simpel gasstrøm gennem opsætning på en kraftig mikroskop. En microincubation kammer anvendes sammen med nitrogengas strømme gennem og en roterende skive konfokalt mikroskopat skabe et kontrolleret miljø, hvor dyrene kan blive afbildet in vivo. Brug af GFP-mærkede gamma tubulin og histon, kan den dynamik og anholdelse af celledeling monitoreres før, under og efter udsættelse for en ilt-frataget miljø. Resultaterne af denne teknik har en høj opløsning, detaljerede videoer og billeder af cellulære strukturer i blastomeres af embryoner udsat for ilt afsavn.

C. elegans embryoner udsat for svær iltmangel (iltmangel) er i stand til at overleve ved at arrestere biologiske processer, herunder udvikling og celledeling 7. Den anoxia-induceret arrest af celledeling kan overvåges ved en subcellulære niveau, ved anvendelse af en microincubation kammer sammen med nitrogengas strømme gennem og en roterende skive konfokalt mikroskop for at skabe et kontrolleret miljø, hvor dyrene kan blive afbildet in vivo (fig. 1). Cellulære struktur...

Watch this Scientific Journal Video about Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos at JoVE.com

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

Beskrevet her er en<em&gt; In vivo</em&gt; Teknik til billede subcellulære strukturer i dyr udsat for anoxi ved hjælp af en gasstrøm gennem microincubation kammer i forbindelse med en roterende skive konfokalt mikroskop. Denne metode er enkel og fleksibel nok til at passe til en række af eksperimentelle parametre og modelsystemer.

Brug af Time Lapse Microscopy at Visualiser Anoxia-induceret Suspended Animation i<em&gt; C. elegans</em&gt; Embryoner

Caenorhabdits elegans has been used extensively in the study of stress resistance, which is facilitated by the transparency of the adult and embryo stages ...

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Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

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