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.
- Theoviductswiththeupperpartoftheuterusattachedareremovedfrom2.5dayspost-coitum(dpc)superovulatedCD-1femalesandplacedintoadropofM2.
- Underdissectingmicroscopetheoviductsareflushedbyinsertingtheflushingneedleattachedtoa1mlsyringeofM2intotheinfundibulum.
- TheembryosarecollectedusingmouthorfingercontrolledpipetteandwashedthroughseveraldropsofM2mediumtoremoveanydebris.
- 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.
- Passthegroupof20-25embryosthroughthedropofmannitolandplacethembetweenelectrodesonebyone,leavingdistancebetweenthem.Mostembryoswillproperlyorient.Pickupthosewhicharenotorientedandplacethemrightmanually.
- Whenalltheembryosareproperlyorientedpushthetriggertoapplythepulse.
- TransfertheembryosintothenewM2drop.
- Repeatsteps5-7withnewgroupsofembryos.
- RinseembryosthroughKSOMdropsandplacethemintomicrodropsundertheoilintheincubator.
- 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.
- PlacefewrowsofKSOMmicrodrops(roughly3mmindiameter)intotissueculturedishusingsyringe(e.g.3dropsinthefirstandfourthand4-5inthesecondandthirdrows),coverwithoil.
- Sterilizeaggregationneedlebywashinginethanol.
- Makesixormoredepressionsineachmicrodrop(leavingafewdropsintactforEScellselection.
- Keeptheplateat37oC,5%CO2.
RemovalofZonaPellucida
- Dissectingmicroscope;
- AcidTyrode"s(e.g.Sigma:T1788);
- SterileplasticPetridishes(100x15mm);
- M2andKSOMmediumin1mlsyringes;
- 9""Pasteurpipettes;
- MouthorFingercontrolledpipette.
- PlaceafewdropsofM2,KSOMandacidTyrode"sinthePetridish.
- WashthegroupofembryoswithaslittlemediumaspossIBLethroughonedropofAcidTyrode"s,thentransfertoafreshdropofthesamesolution.
- Observezonadissolution.
- ImmediatelytransfertheembryosintoadropofM2mediumassoonasthedissolutioniscompleted.
- WashtheembryosatleasttwiceinKSOMdropsbeforeputtingthemintheaggregationplate.
- Transferembryosintoaggregationplatebyplacingthemonebyonebesideeachdepressionaswellasinsideeachdepression(twoembryoswillbeusedtoform"sandwich"withES-cellsclump).ItispossibletouseonlyonetetraploidembryotoaggregatewithES-cellsbutsuchaggregateswillneedanadditionalnightofculturetoformblastocysts.
- PrepareES-cellsforaggregationbythattime.
EScells/tetraploidembryo"SANDWICH"aggregation
- Dissectingmicroscope;
- Preparedaggregationplatewithdepressionsandembryoswithremovedzona;
- TrypsinizedES-cells;
- MouthorFingercontrolledpipette;
- 9""Pasteurpipette.
Method:
- ChooseclumpsoflooselyconnectedEScellsandtransferthemintomicrodrops(notcontainingembryos)ofaggregationplateforfinalselection.
- SelectfewclumpsofEScells(8-15cellseach);placeeachinadepressionnexttoanembryo.
- PickupthecorrespondingembryooutsidethedepressionandplaceitonthefreesideoftheEScellclump,forming"sandwich".
- 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);
- 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.
