Separation of sea urchin blastomeres
来自 : 蚂蚁淘
Objective:toseparatetheBlastomereatdifferentpointsindevelopmentandobservethegrowthoftheresultingembryos.Procedure:Weextractrfthegametesfromtheseaurchins,fertilizedtheseaurchineggs,andremovedthefertilizationenvelope.Thecellswerethenbeallowedtocleave,atwhichpointtheywerebeintroducedintoahypertonicseawatersolution.Thiscausedthecellstoshrinkandbreakapart.Oncethemajorityofthecellshadseparated,thewaterwasreplacedwithnormalseawater.Theirgrowthwasbecomparedtothatofthecontrolgroupthatwasnotdivided.
- TheeggswerewashedseveraltimeswithASWbyallowingthemtosettleandpouringoffthewater.Thiswillremovedsomeofthejellycoat.
- Enougheggstocoverthebottomofa50mlbeakerweretransferedalongwith10mlof10mMp-aminobenzoicacidinASW.
- 50mlofspermweredilutedin5mlofASW.2dropswereusedtofertilizeeggs.
- Thepresenceofthefertilizationenvelopewascheckedafter5and10minutesinthecontrolculture.
- Theeggswerepouredthroughanylonfiltermountedtotheendofa50mlbeaker.Thiswasrepeateduntilmostofthefertilizationenvelopeshavebeenremoved.
- Thecellswereallowedtosettleforafewminutes.
BlastomereSeparation
- Thecellswerethenseparatedinto3100mlbeakersfullofASW.Carewastakensoasnottoaddtoomuchoftheseawater-PABAsolution.
- Wethenwaitedforthecontrolculturetocleaveforthefirsttime.
- Thenmostofthewaterwaspouredfromthefirstexperimentalculture.
- Thebeakerwasgentlyfilledwithhypertonicseawater.Theculturewasthencheckedafter5minutestoseewhetherosmoticshrinkagecausedthecellstoseparate.Thesolutionwasreplacedwithadditionalhypertonicseawateriftheyhadnotseparated.
- Wewaited10minutesafterthecellsshowosmoticshrinkage.
- Thehypertonicsolutionwaspouredoffandreplaceditwithnormalseawater.Thiswasrepeated.
- Steps3-6wererepeatedforthesecondexperimentalcultureoncethecontrolgroupcleavedathirdtime.
- Theembryoswereallowedtodevelopandobservedatdifferentstagesintheirgrowth.
ResultsWhilethecontrolembryosgrewtoformnormalpluteustheexperimentalembryosdidnotcleaveonceseperatedfromeachother.Thecontrolorganismswerestainedwith5C7,EctoV,andIg8tolookattheirformationeventhoughtherewerenoexperimentalclonestocomparetothem.Anotherprocedureinvolvingacalciumpoorseawatersolutioncanbeusedtoseperatetheembryosandmayproducemoredesirableresults.Clickonthumbnailstoseefullsizepictures.Usethebackbuttonofyourbrowsertoreturntothispage
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