Name: planarian immobilization, partial irradiation, and tissue transplantation
Uploaded: 2012-08-06T12:00:00
Description: Watch this Scientific Journal Video about Planarian Immobilization, Partial Irradiation, and Tissue Transplantation at JoVE.com

Forfatterne vil gerne takke Chiyoko Kobayashi og Kiyokazu Agata for gode råd om planarian transplantation samt tidligere og nuværende medlemmer af Sánchez laboratoriet for uvurderlige diskussioner i udviklingen af ​​disse teknikker. Dette arbejde blev støttet af NIH Training Grant (5T32 HD0791) til OCG og NIH R37GM057260 til ASA. ASA er en Howard Hughes Medical Institute Investigators.

Bemærk: denne protokol foreslår brugen af potentielt farlige materialer (bly og chloretone). Acquire, læse, og følg MSDS for alle potentielt farlige materialer. 1. Animal Kultur, udvælgelse og forberedelse <ol><li> For dyr kultur og håndtering brug planarian vand (1X Montjuic salte 9) og plast overførselspipetter. </li><li> Seksuel biotype Schmidtea Mediterranea kan bruges når den er hævet i laboratoriet under normale dyrkningsbetingelser ...

<ol>
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Vigtigheden af ​​immobilisering Immobilisering er langt den mest kritiske skridt for en vellykket gennemførelse af nogen af ​​disse processer. Hvis planaria ukorrekt immobiliseres forud for delvis bestråling, kan de bevæge sig under blyafskærmning, der producerer usammenhængende og confounding resultater. Desuden, hvis dyrene utilstrækkeligt immobiliseret efter transplantation, vil værtens ormen sandsynlige bevæge sig bort fra transplantatet væv, hvilket resulterer i en fejl i ...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalog nummer </td></tr><tr><td> General Purpose Transfer Pipette </td><td> Samco </td><td> 691 </td></tr><tr><td> Kapillærrør (ID 0,75 mm) </td><td> FHC </td><td> 30-30-0 </td></tr><tr><td> Kapillærrør (OD 0,7 mm) </td><td> FHC </td><td> 30-50-08 </td></tr><tr><td> Parafilm M </td><td> VWR </td><td> 52858-076 </td></tr><tr><td> Kimwipes 34155 </td><td> VWR </td><td> 500029-891 </td></tr><tr><td> Sort filtrerpapir </td><td> Schleicher &amp; Schuell </td><td> 10310809 </td></tr><tr><td> Whatman # 2 filterpapir 1002-055 </td><td> Fisher Scientific </td><td> 09-810B </td></tr><tr><td> Whatman # 3 filterpapir 1003-185 </td><td> Fisher Scientific </td><td> 09-820E </td></tr><tr><td> Cigaretrulning papir </td><td> Zig-Zag, original </td><td> NA </td></tr><tr><td> Petrifade </td><td> VWR </td><td> 82050-544 </td></tr><tr><td> Tang DUMONT, INOX # 5 </td><td> FST </td><td> 11251-20 </td></tr><tr><td> Chloretone </td><td> Sigma Aldrich </td><td> 112054 </td></tr><tr><td> Kasein </td><td> Sigma Aldrich </td><td> C3400 </td></tr><tr><td> Bly Shields </td><td> Alpha Systems Corp Bluffdale, UT </td><td> Custom design </td></tr><tr><td> XRAD-320 Biologisk strålingsovnen </td><td> Precision X-Ray, North Branford, CT </td><td> NA </td></tr></tbody></table>

planarian immobilization, partial irradiation, and tissue transplantation

Det planarian, en ferskvands fladorm, har vist sig at være et effektivt system til dissekere metazoan regenerering og stamcellebiologi 1,2. Planarian regenerering af eventuelle manglende eller beskadigede væv er gjort mulig ved voksne stamceller kaldes neoblasts 3. Selv om disse stamceller er blevet definitivt vist at være pluripotente og enkeltvis stand til at rekonstituere et helt dyr 4 heterogeniteten i stamcellepopulation og dynamikken i deres cellulære adfærd stort set uløst. Grund af det store antal og udbredelse af stamceller gennem planarian kroppen plan, der avancerede metoder til manipulering subpopulationer af stamceller for molekylær-og funktionelle studier in vivo brug. Vævstransplantation og delvis bestråling er to metoder, som en subpopulation af planarian stamceller kan isoleres til yderligere undersøgelse. Hver teknik har forskellige fordele. Tproblem transplantation tillader indføring af stamceller, i en naiv vært, der er enten iboende genetisk forskellige, eller tidligere er blevet behandlet farmakologisk. Alternativt delvis bestråling muliggør isolering af stamceller i en vært, anbragt på væv uden stamceller, uden indføring af et sår eller en bundstykket i væv integritet. Ved hjælp af disse to metoder, kan man undersøge celle autonome og ikke-autonome faktorer, der styrer stamceller funktioner, såsom proliferation, differentiering og migration. Både vævstransplantation 5,6 og partiel bestråling 7 har været anvendt historisk til at definere mange af de spørgsmål om planarian regenerering, der fortsat er under undersøgelse i dag. Imidlertid har disse teknikker forblev underudnyttede grundet arbejdskrævende og usammenhængende art tidligere metoder. De protokoller, der præsenteres her repræsenterer et stort skridt fremad i begrænse den tid, ennd nødvendige indsats for at reproducerbart frembringe store antal podede eller delvist bestrålede dyr med effektiviteter nærmer sig 100 procent. Vi dækker kultur store dyr, immobilisering, forberedelse til delvis bestråling, vævstransplantation, og optimering af animalsk opsving. Endvidere arbejdet beskrevet her, viser den første anvendelse af den delvise bestråling fremgangsmåde til brug med den mest undersøgte planarian, Schmidtea Mediterranea. Derudover effektiv vævstransplantation i planaria åbner døren for den funktionelle test af delpopulationer af naive eller behandlet stamceller i genindsættelse assays, som længe har været guld-standard metode til analyse voksne stamceller potentiale i pattedyr 8. Bred indførelse af disse teknikker vil uden tvivl føre til en bedre forståelse af de cellulære adfærd af voksne stamceller under vævshomeostase og regenerering.

Watch this Scientific Journal Video about Planarian Immobilization, Partial Irradiation, and Tissue Transplantation at JoVE.com

Planarian Immobilization, Partial Irradiation, and Tissue Transplantation

En effektiv fremgangsmåde til podning væv veldefineret og ensartet størrelse mellem planaria er beskrevet. Også indbefattet er en beskrivelse af, hvordan immobilisering anvendte teknik til transplantation kan tilpasses, sammen med bly skjolde, til delvis bestråling af levende dyr.

Planarian Immobilisering, Delvis bestråling og vævstransplantation

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian ...

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