基因突变

Axolotl Protocol: Transplantation of the Gill Organ Field

Objective:Theoverallobjectiveofthislaboratoryexperimentistoexaminespecificationintheaxolotlthroughtransplantationofthegillorganfield.

Introduction:Theaxolotl(Ambystomamexicanum)isalargesalamanderwhichisnativetoLakeXochimilco,Mexico.Theaxolotlisamemberofalargergroupofsalamandersknownasmolesalamanders.Othermembersofthisgroupincludetigersalamandersandspottedsalamanders.Thewildtypeaxolotlisdistinguishedbyitsdarkcolorwhichmaycontaingreenishmottlingandsilverypatches.Thealbinotypeaxolotliswhite(withoutpigment).Theaverageaxolotlcanreachalengthofapproximately12inchesfromnosetotailandcanweighupto300grams.Axolotlsareneotenic,whichmeanstheywillpossesstheirfeatheryexternalgillsandtailfintheirentireliveswhiletheymaintainanaquaticlifestyle

Background:Thegoalofthisexperimentistodetermineifthegillorganfieldisspecifiedattheparticulartimethatitistransplanted.AccordingtoGilbert(2000),acelloratissueissaidtobespecifiedwhenitiscapableofdifferentiatingasitwouldhavewhenplacedinaneutralenvironmentsuchasapetridishortesttubeoraftertransplantationtoanectopicsite.Anorganfieldisalargeregionoftheembryoinwhichcellsarespecifiedgenerally(leg,gill)butarenotspecifiedforaspecificorgansuchasatoeorfinger.Organfieldsaredevelopedintheaxolotlthroughinduction.Aninteractionatcloserangebetweentwoormorecellsortissuesthathavedifferentpropertiesiscalledinduction(Gilbert,2000).Therearetwocomponentsnecessaryforinductiveinteraction.Thefirstcomponentistheinducer,whichisthetissueorcellsthatproduceasignal(s)thatwillchangethecellularbehaviororactivityoftheothercellsortissue.Thesecondcomponentistheresponder,whichisthetissueorgroupofcellsthatarebeinginduced(Gilbert,2000).Coordinationintheconstructionoforganfieldsisachievedasonegroupofcellschangesthebehaviorofanadjacentsetofcells,whichwillcausethecellstochangeshape,mitoticrate,orfate(Gilbert,2000).Oncetheorganfieldisspecified,inductiveeffectswillhelpfurtherdevelopthefieldintothefinalorgan.Bytransplantingthegillorganfieldfromawildtypeaxolotlintoanalbinoaxolotlandviceversa,weplanonexaminingifthegilldevelopsasitwouldhaveinitsoriginalenvironmentorifitisinfluencedbythesurroundingtissues.Weexpectthegilltodevelopasitwouldhaveinitsoriginalenvironment.

Procedure:1.Obtainaxolotlembryos.Forthisexperiment,wewillbeusingwildtypeandalbinoaxolotlembryosatstage21intheirstagingseries.2.Preparetheaxolotlembryosasfollows:a.manuallydejellyembryoofappropriatestageandkeepinHEPES-bufferedModifiedSteinberg"ssolution(HBSt)containingantibioticsuntiluse.Formicrosurgery,alsoremovemembranearoundembryo.b.placeembryosintooperatingdishcontaining1xHBStandantibioticsc.usecleantechnique;diptools,Pipettes,andglassbridgesin70%EtOHandthenintosterileHBStbeforeusing3.Startmicrosurgeryonyourdonorembryo.Locatethegillorganfieldwhichisinthefirstanteriorthirdoftheembryo(towardhead)andrightabovethesmallcrestorindentionclosertotheventralside(Rugh,1962).Isolatethegillorganfield(ortheregionthatwillbecomethegillorganfield)bycuttingarounditanddetachingthatspecificpieceoftissuefromtheembryo.4.Starttransplantationonyourrecipientembryo.Cutasmallslitinaregionclosetothegillorganfieldontherecipient.Gentlyslipdonorgillorganfieldintoslitandbesuretosecurethegraftinplace.ItiscriticaltotrytogetthegraftasfarunderthecutaspossIBLetoensurethattherecipientwillbegintoincorporatethenewtissueintoitsdevelopmentprocess.

Figure1:

A.A21stagealbinoaxolotlembryowithatransplantedwildtypeaxolotlgraft(supposedlythegillfield).Thepicturewastakenimmediatelyaftermicrosurgery.

B.A21stagewildtypeaxolotlembryowithatransplantedalbinoaxolotlgraft.Thepicturewastakenimmediatelyaftermicrosurgery

5.Oncethesurgeryiscomplete,besuretotakepicturesofanormaldonorandrecipientandthenthenewlytransplantedgraftoftherecipient.Picturesshouldbetakenatvariousstagestoobservedevelopment(tobedeterminedatdiscretionofresearcher).6.TheembryosshouldbeleftinHBStatroomtemperaturetocontinuetodevelopment.Observebothdonorsandrecipientsastimecontinuestoexaminetheeffectofthetransplantedgraft.7.MakeOBSERVATIONSasdevelopmentcontinues...thisisthekeytoobtainingresults!8.Afterthefirstday,itisimportanttoreplacetheoperatingsolutionwith10%modifiedSteinberg"ssolution.Thegraftwilldevelopbetterinalowerconcentrationofcertainchemicals.Todothis,simplypipetteofftheoperatingsolutionandpourinthe10%modifiedSteinberg"ssolution.

Results/Discussion:Graftsweresuccessfullytransplantedintodonorrecipients.AwildtypeaxolotlthathadreceivedanalbinotransplantsurvivedthelongestanddisplayedtheABIlitytoretainthetransplantedgraftandcontinuedevelopment.Thealbinorecipientsofwildtypegraftswerenotassuccessfulbecausethegraftblendedintothealbino"snaturalcolorandcouldnotbeeasilydistinguished.Microsurgerywascomplicatedandtheregionsthatweretransplantedeitherwerenotthegillorganfieldorwerenotdirectlytransplantedintothegillorganfield.However,itisimportanttonotethatthosegraftsthatweretransplanteddidsuccessfullystayintherecipientsandtherecipientscontinuedtodevelopwiththegraft.

Figure2:Apictureofawildtypeaxolotlembryo2daysafterthealbinoaxolotlgrafthadbeentransplantedintoit.Thegraftislocatedbelowthegillfieldbutstillhasbeensuccessfullytransplantedandhasbecomeapartofthedevelopingembryo.

Weexpectedthatthetransplantedgraftwouldhavedevelopedasifitwereapartitsoriginalenvironment.Thisimpliesthatthegillwouldhavebeenspecifiedpriortotransplantation.Upontransplantation,inductiveeffectswouldallowthetransplantedgrafttoeventuallyformthegill.Therearetworeasonswhywemightnothaveexaminedanyofourexpectedresults.Thefirstisthepossibilitythatthegillorganfieldhadnotbeenspecifiedpriortotransplantation,whichdidnotallowittodevelopproperlywhenitwastransplantedintotherecipients.Thesecondreasonisduetotechnology.Thiswasaverycomplicatedexperimentthatrequiredskilledmicrosurgeryonembryos.Sincethiswasafirstattemptatmicrosurgery,ourtechniqueandtechnologymighthavebeeninsufficientwhichcausedpoorresultstobeobtained.

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