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PRODUCTION OF ES CELL CHIMERAS BY AGGREGATION WITH EIGHTCELL S...

蚂蚁淘  /发布时间:1545760852 /浏览量:195   

Thetechniquedescribedhereisaslightmodification(March1997)ofmethodspresentedin:NagyA.,J.Rossant.1993.ProductionofcompletelyEScell-derivedfetuses.In:GeneTargeting:APracticalApproach.(ed.A.Joyner).IRLPressatOxfordUniversityPress.

Recoveryof2-cellstageembryos

Recoveryof2-cellstageembryosisverysimilartothatofthe8-cellstageembryosanditmustbedoneontheday1.5dpc.Itissafertocollectlate2-cellsstageembryostoavoidtheso-calledtwo-cell-stageblock.Thepresenceof10-15%3-4-cellstageembryosamong2-cellindicatestheappropriatetiming.Theflushing46+hoursafterhCGinjectionisrecommended.

  • 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.

Productionoftetraploidembryos

Fusionoftheblastomeresof2-cellstageembryosoccurswhenasquarepulseisappliedperpendiculartotheplaneofcontactofthetwocells.Thepulseparametersvarrydependingontheelectrodesandpulsegenerator.WeuseCell-fusioninstrument,CF-150BavailablefromBLSLtd.,Hungarywithfollowingparameters(fornon-electrolytefusion):Voltage-30V;Duration-35microsec;Numberofpulses-2;AdjustableACfieldisappliedtoallowthecorrectorientationofembryos(enable,1or2Vonthedisplay).ToohighanACfieldcancauselysis.

  • CF-150Bcell-fusioninstrumentwithanelectrodechamber(Thecompanymanufacturethreekindsofelectrodechambers:GSS-250,GSS-500orGSS-1000,whichdifferinthegapdistance.Allworkwellfortetraploidproduction.Thechoiceisbasedonpersonalpreference.)
  • twodissectingmicroscopes;
  • M2andKSOMmedia;
  • 0.3MMannitol(SigmaM4125)(dissolvedinultrapurewaterwithadded0.3%BSA(SigmaA4378)andfilteredthrough0.22micronMilliporefilter,storedinaliquotsat-20C);
  • tissueculturedishwithKSOMmicrodropscoveredwithoil(SigmaM8410);
  • mouthorfingercontrolledpipette;

Method:

1.TurnontheCF-150Bpulsegenerator.Makesureyousettheswitchonthebacksideofthemashineto"Non-electrolyte".DoNOTusethe"Normal"position!

2.Puta100mmPetridishcontainingtheelectrode-chamberunderadissectingmicroscope,connectthecablestothepulsegeneratorandadjustallparametersbysettingthe<mode>buttontoVoltageandturningtheappropriatedialontheleftside,thensettingthe<mode>todurationandturningtheappropriatedialuntilthedesirednumberisdisplayed.

TypicalsettingsforGSS-250:40V,30microsec,2repeats,forGSS-500:75V,35microsecandforGSS-1000:137V,26microsec.Theparametershowevercoulddependonlocalconditionsandembryosused.Theyshouldbeoptimizedempirically.

Onthefrontpanel,inthelowerrightcorner,youhaveatwobuttonsandadial.Pushthefirstbutton,whichiscalled"HFSINUS"-"ENABLE"untilthediodlitsupanditissoenabled.Pushthebuttonontheothersideofthedialuntilalsothisisenabled.Thisbuttoniscalled"ATTENUATOR"-"AMP/10.Nowturnthedialuntil1.0-2.0isshownonthedisplay.Whichstrengthyoushouldchoosedependsonhowquicklyyoucanwork,andthesensitivityofyourembryos.Thehigherthisvalue,thefasterwilltheembryosalignintherightposition,butatoohighvalueformorethen20-30secondssecondswillharmthem.Thisyoushouldoptimizeempirically.

3.PlacetwolargedropsofM2mediumandtwodropsofmannitolsolutioninthesecond100mmPetridishunderotherdissectingmicroscope.Placelargedropofmannitoloverelectrodes.

4.Place50-100embryosinonedropofM2.Thenumberofembryosdependsonthespeedsincethedropofmannitolovertheelectrodechambercannotbeusedforlongerthat10-15minutes(itevaporatesandthefusionbecomeslessefficient)andshouldbechangedtofreshoneafterthattime.

  1. Passthegroupof20-25embryosthroughthedropofmannitolandplacethembetweenelectrodesonebyone,leavingdistancebetweenthem.Mostembryoswillproperlyorient.Pickupthosewhicharenotorientedandplacethemrightmanually.
  2. Whenalltheembryosareproperlyorientedpushthetriggertoapplythepulse.
  3. TransfertheembryosintothenewM2drop.
  4. Repeatsteps5-7withnewgroupsofembryos.
  5. RinseembryosthroughKSOMdropsandplacethemintomicrodropsundertheoilintheincubator.
  6. Repeatsteps4-9withtherestofembryos.

Fusionofblastomeresshouldbecompletedin20-40minutes.Sinceembryosarerecoveredatthelate2-cellstage,thesecondmitoticdivisionisexpectedsoonafterfusion.Thereforeitisimportanttoselectfortheperfectlyfusedtetraploidembryos20-60minafterapplicationofthepulse.Itissafesttotransferthetetraploidsintoanewculturedishornewmicrodrop.Underoptimalconditionstherateofunfusedandlysedembryosdoesnotexceed5%.Thetetraploidembryosareculturedovernight.Majorityofthemformthe2-cellstage.Bynoonofthenextdaymostofthemhavecleavedoncemoreandreachedthe4-cellstage.Thisstageisequivalenttothediploid8-cellstageandshouldbeusedforaggregationwithES-cells,tetraploidembryosstartcompactingatthisstage.Someembryosarestillatthe2-cellstagethenextstage,theyaredelayed.Dependingonthetotalnumberof4-cellstageembryosandnumberofrecipients,2-cellstagemightstillbeusedforaggregationsbutwithlimitedsuccess.

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.
  4. Keeptheplateat37oC,5%CO2.

RemovalofZonaPellucida

  • Dissectingmicroscope;
  • AcidTyrode"s(e.g.Sigma:T1788);
  • SterileplasticPetridishes(100x15mm);
  • M2andKSOMmediumin1mlsyringes;
  • 9""Pasteurpipettes;
  • MouthorFingercontrolledpipette.
  1. PlaceafewdropsofM2,KSOMandacidTyrode"sinthePetridish.
  2. WashthegroupofembryoswithaslittlemediumaspossIBLethroughonedropofAcidTyrode"s,thentransfertoafreshdropofthesamesolution.
  3. Observezonadissolution.
  4. ImmediatelytransfertheembryosintoadropofM2mediumassoonasthedissolutioniscompleted.
  5. WashtheembryosatleasttwiceinKSOMdropsbeforeputtingthemintheaggregationplate.
  6. Transferembryosintoaggregationplatebyplacingthemonebyonebesideeachdepressionaswellasinsideeachdepression(twoembryoswillbeusedtoform"sandwich"withES-cellsclump).ItispossibletouseonlyonetetraploidembryotoaggregatewithES-cellsbutsuchaggregateswillneedanadditionalnightofculturetoformblastocysts.
  7. PrepareES-cellsforaggregationbythattime.

EScells/tetraploidembryo"SANDWICH"aggregation

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

Method:

  1. ChooseclumpsoflooselyconnectedEScellsandtransferthemintomicrodrops(notcontainingembryos)ofaggregationplateforfinalselection.
  2. SelectfewclumpsofEScells(8-15cellseach);placeeachinadepressionnexttoanembryo.
  3. PickupthecorrespondingembryooutsidethedepressionandplaceitonthefreesideoftheEScellclump,forming"sandwich".
  4. Assembleallaggregatesinthismanner,checktheplate,andcultureovernightat37oC,5%CO2.

Thefollowingafternoon,themajorityofaggregatesshouldhaveformedblastocysts.Atthistimetheyshouldbetransferredintotheuterus

of2.5dpcpseudopregnantfemales.Wetransferamaximumof8-10embryosintoeachuterinehornofapseudopregnantrecipient.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.ProductionandanalysisofchimericmiceinTeratocarcinomasandEmbryonicStemcells:aPracticalApproach(ed.E.J.Robertson)IRLPress,Oxford,Washington,D.C.3.Pappaioannou,V.,R.Johnson.1993.ProductionofchimerasandgeneticallydefinedoffspringfromtargetedEScells.InGeneTargeting:APracticalApproach(ed.A.Joyner)IRLPressatOxfordUniversityPress4.StewartC.L.1993.ProductionofChimerasbetweenEmbryonicStemCellsandEmbryos.inMethodsinEnzymology.vol.225.AcademicPressInc.

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