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PRODUCTION OF ES CELL CHIMERAS BY AGGREGATION WITH EIGHTCELL STAGE EMBRYOS188bio精品生物—专注于实验室精品爆款的电商平台 - 蚂蚁淘旗下精选188款生物医学科研用品
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PRODUCTION OF ES CELL CHIMERAS BY AGGREGATION WITH EIGHTCELL STAGE EMBRYOS

Introduction

Thetechniquedescribedhereisaslightmodification(March1997)ofmethodspresentedin:NagyA.,J.Rossant.1993.ProductionofcompletelyEScell-derivedfetuses.InGenetargeting:aPracticalApproach.(ed.A.Joyner).IRLPressatOxfordUniversityPress.ToproducecompletelyEScell-derivedembryosaclumpofEScellsissandwichedbetweentwotetraploidembryos.ForEScellaggregationchimeraswithdiploidembryos,oneembryoisaggregatedwithaclumpofEScells.Eveninthiscase,theEScellinternalizationisefficientenoughtoachieveahighlevelofEScellcontribution.Thebenefitofsingleembryoaggregationistwofold:First,doublethenumberofaggregatescanbeproducedinasingleexperiment.Second,wehavefoundthattheefficiencyofgermlinetransmissionofthetargetedalleleisbetterthanwiththesandwichtype.Thisobservationmaybeexplainedasfollows.Whenmale(XYgenotype)EScellsareaggregatedwithafemale(XXgenotype)morula,resultinginaphenotypicallymale,onlyprimordialgermcellsderivedfromtheXYEScellscancontributetothefunctionalgametes.TheXXprimordialgermcellsfromthehostembryowillnotformgametes,thereforefavoringthegermlinecontributionofEScellscontainingthedesiredmutantallele.Thisarrangement(XXhostmorula,XYEScells)canoccurin50%ofthesingleembryoaggregations,butonlyin25%ofthesandwichtypeaggregations.

Flushingofeight-cellstageembryos

  • Dissectingmicroscope;
  • Flushingneedle(ThesharptipofNo.30G1/2needleiscutoffandthenroundedusingsharpeningstone);
  • 1mlsyringe;
  • Dissectinginstruments(fine-pointedscissors,fineforceps);
  • No.5forceps(Dumont);
  • MouthPipette(aspiratormouthpiece,latextubing,bluetip)alternatively:
  • Fingercontrolledpipette(Manostattubing,yellowtip,scalpelblade);
  • 9""Pasteurpipettes;
  • 70%Ethanol;
  • AlcoholburnerorBunsenburner;
  • SterileplasticPetridishes(100x15mm);
  • SterileOrganCulturedishes(60x15mm,Falcon,3037);
  • M2andKSOMmedia.

1.Theoviductswiththeupperpartoftheuterusattachedareremovedfrom2.5dayspost-coitum(dpc)superovulatedCD-1femalesandplacedintoadropofM2.2.Underdissectingmicroscopetheoviductsareflushedbyinsertingtheflushingneedleattachedtoa1mlsyringeofM2intotheinfundibulum.3.TheembryosarecollectedusingmouthorfingercontrolledpipetteandwashedthroughseveraldropsofM2mediumtoremoveanydebris.4.TheembryosarewashedinKSOMmediumandculturedinorganculturedishinKSOMat37oC,5%CO2.

Preparationofaggregationplate

  • Dissectingmicroscope;
  • Steriletissueculturedishes(EasyGrip35x10mm,Falcon3001-3);
  • 1mlsyringewith26G1/2needleofKSOMmedium;
  • lightmineraloil(EMBRYOTESTED)(e.g.Sigma:M8410);
  • aggregation(darning)needle(DN-09,BLSLtd.,Hungary,)
  • 70%ethanol.

1.PlacefewrowsofKSOMmicrodrops(roughly3mmindiameter)intotissueculturedishusingsyringe(e.g.3dropsinthefirstandfourthand4-5inthesecondandthirdrows),coverwithoil.2.Sterilizeaggregationneedlebywashinginethanol.3.Makesixormoredepressionsineachmicrodrop(leavingafewdropsintactforEScellselection)bypressingthedarningneedleintotheplasticandmakingslightcircularmovement.Donottwisttheneedle.Thismovementcreatesatinydepressionwithclearsmoothwall.Itshouldbedeepenoughtoholdtheembryo.4.Keeptheplateat37oC,5%CO2.

RemovalofZonaPellucida

  • Dissectingmicroscope;
  • AcidTyrode"s(Sigma:T1788);
  • SterileplasticPetridishes(100x15mm);
  • M2andKSOMmediumin1mlsyringes;
  • 9""Pasteurpipettes;
  • MouthorFingercontrolledpipette.

1.PlaceafewdropsofM2,KSOMandacidTyrode"sinthePetridish.2.WashthegroupofembryoswithaslittlemediumaspossIBLethroughonedropofAcidTyrode"s,thentransfertoafreshdropofthesamesolution.3.Observezonadissolution.4.ImmediatelytransfertheembryosintoadropofM2mediumassoonasthedissolutioniscompleted.5.WashtheembryosatleasttwiceinKSOMdropsbeforeputtingthemintheaggregationplate.6.Transferembryosintoaggregationplatebyplacingthemonebyonebesideeachdepression,alternativelyplaceoneembryoinsideeachdepression.7.PrepareES-cellsforaggregationbythattime.

EScells/embryoaggregation

Dissectingmicroscope;Preparedaggregationplatewithdepressionsandembryoswithremovedzona;TrypsinizedES-cells;MouthorFingercontrolledpipette;9""Pasteurpipette.

1.ChooseclumpsoflooselyconnectedEScellsandtransferthemintomicrodrops(notcontainingembryos)ofaggregationplateforfinalselection.2.SelectfewclumpsofEScells(8-15cellsineach);placeeachclumpinadepressioninthemicrodropcontainingembryos.3.Pickupthecorrespondingembryoandplaceitontheclump,alternatively,iftheembryoisalreadyinsidethedepression,placetheclumpofcellsnexttoit.4.Assembleallaggregatesinthismanner,checktheplate,andcultureovernightat37oC,5%CO2.Thefollowingmorning,themajorityofaggregatesshouldhaveformedblastocysts.Wetransferamaximumof8-10embryosintoeachuterinehornofa2.5dpcpseudopregnantrecipient.MatureCD-1femalesareusedaspseudopregnantfostermothersandorderedataweightof30+g.Intheeventofarecipientshortage,itispossible:

  • totransferupto24-26embryosperrecipient;
  • toculturetheaggregates(preferablymorulae)foronemoredayandtransfertheminto2.5daypseudopregnantfemales;
  • touse3.5daypseudopregnantfemales.

Transferofembryos

  • 2.5dpcpseudopregnantfemales;
  • Instruments:
  • Scissors;
  • Semkenforceps(straightorcurvedwithserratedtips);
  • Forcepswith1x2teeth;
  • Dumontss/mcorNo.5forceps;
  • Serrefine(e.g.FineScientificTools:18050-28or8051-28);
  • 26G1/2or30G1/2needle;
  • Autoclipapplier(ClayAdamsB-D763007);
  • Autoclips(ClayAdamsB-D7631);
  • Mouthcontrolledpipette;
  • M2medium;
  • 2.5%Avertin:
    • 2,2,2,-Tribromethanol2.5g(Morre-TecIndustries#1693);
    • Tert-amylalcohol5.0ml(Fisher:A-730-1).

      DissolvetribromethanolinTert-amylalcohol,thenaddto200mldistilledwater.Placeonamagneticstirreruntilsolutionisinonephase.Storeinbrownbottleandkeeprefrigerateduntiluse.Shouldbewarmedandshakenbeforeuse.Dosageis0.2ml/10gbodyweight.

Theembryotransferprocedureisdescribedindetailsinmanypublications,suchasthefollowing:1.Hogan,B.,F.Constantini,E.Lacy.1986.ManipulatingtheMouseEmbryo.ColdSpringHarbor,NewYork.2.Bradley,A.1987.Productionandanalysisofchimericmice.InTeratocarcinomasandEmbryonicStemCells:APracticalApproach(ed.E.J.Robertson)IRLPress,Oxford,Washington,D.C.3.Pappaioannou,V.,R.Johnson.1993.ProductionofchimerasandgeneticallydefinedoffspringfromtargetedEScells.InGeneTargeting:APracticalApproach(ed.A.Joyner)IRLPressatOxfordUniversityPress.4.StewartC.L.1993.Productionofchimerasbetweenembryonicstemcellsandembryos.InMethodsinEnzymology.vol.225.AcademicPressInc.


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