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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