PurposeandBackgrounds

CHOlec3.2.8.1cells

CHOLec3.2.8.1cellshavefourindependentmutationsintheN-andO-glycosylationpathways(Stanley,1989).N-linkedcarbohydratesproducedbyCHOLec3.2.8.1cellsareallofthehighmannosetype,butdifferinthenumberofmannoses,rangingfromMan₉toMan₅.O-glycosylationishomogenous,withonlyasingleGalNAcresidueattachedpersite.Whenculturedinthepresenceofthealpha-glucosidaseIinhibitorN-butyl-deoxynojirimycin(NB-DNJ),glycoproteinsproducedinCHOLec3.2.8.1cellsarealmostcompletelysusceptIBLetoEndoHdigestion(Davis,1995;Ikemizu,1999).EndoHcleaveschitobiose,leavingasingleN-linkedN-acetylglucosaminepersite,whichisidealformaintenanceofproteinsolubilityandspecialcarbohydrate-proteininteractions,suchasbetweenthefirstN-acetylglucosamineresidueandtryptophan.(Onintactproteins,EndoHcleaves(Man)_5-9chainsbut(Man)_3-4chainsareresistant.)

Vectors

UseexpressionvectorswithstrongpromotersuchasimmediateearlyCMV(pCDNA3.1etc.)orhumanEF-1a(pEF1/V5-Hisetc.).pBJ-5/GSvectoralsogivesyouhighexpressionbutrequiresadditionalcloningstrategy.

Materials

Medium

Sigmaprovidesspecialserum-freemediumforCHOcells(C-1707).Addglutamine,nonessentialaminoacids,penisilin/streptomycin,and10%FCS.FCScanbeomittedbutswitchingmustbedoneoveracoupleofmediumchanges(i.e.,changeto5%FCSonceandto2%twicefollowedbycompletelyswitchingtoserum-freemediumetc.).InthecaseofSigma"smediabeingdiscontinued(whichhappenedbefore),youcanpreparebasicmedium(J/J)byyourselfbutthiscannotbeusedasserum-freemedium.

Recipefor10ltofJ/Jmedium:To9ltofmili-Qwater,add

1. MEMw/oGln(Gibco,#11700-077).Pwdrfor10lt.NaHCO₃,27.75gglucose:30gnucleosides:adenosine(SigmaA-4036),70mg;guanosine(SigmaG-6264),70mg;cytidine(SigmaC-4654),70mg;uridine(SigmaU-3003),70mg;thymidine(SigmaT-1895),24mgglutamicacid(G-1626)andasparagine(A-0884);600mgeachsodiumpyruvatesolution(100X,100mM,GIBCO#11360-021),100mlnonessentialaminoacidsolution(100X,10mM,GIBCO#11140-019),200ml
2. Peniciline/Streptomycin(100x),100ml

AdjustpHto7.15andfiltersterilize.

SelectionMarker

CHOLec3.2.8.1cellsarerelativelyresistanttoneomycin(GENETICING418;GIBCO#11811-031).Itisbettertodrawkillcurveforeachbatchofcells(andG418)everytimeyoudotransfection,butgenerallyyoucanstartwith1mg/ml.Evenwiththishighconcentration,itwilltakemorethanaweektokillallofthenon-transfectedcells.Puromycin(Sigma,P-7255)killsCHOLeccellswithinacoupleofdaysatarangeof4-8µg/ml,usuallyuse10µg/ml.Alwaysincludenegativecontroltransfection(electroporationwithoutDNA)toknowwhetherantibioticsworksOK.

Others

• ElectroporationPBS(EPBS):NaH₂PO₄•H₂O96.6mg;Na₂HPO₄•7H₂O482.4mg;NaCl2.197gin250mlwater,sterilefilteredElectroporationcuvette(4mmgap,BTX#01-000196-01)
• 96-welltissuecultureplate(lowevaporation)

Procedure

Day-1

SplitconfluentCHOLeccellsinto75cm2flasks(1/3dilution).

Day0

PreparationofDNA

1. take10µgDNA/constructadd1/10vol3MNaOAc(pH5.2),mixadd2Xvol100%EtOH,mixplaceondryice,5-10minSpin10minatmaxspeedRemovesupernatan
2. Washpelletwith70%EtOH

Electroporation

1. Cellsshouldbe50~70%confluent.Detachcellswithtrypsin/EDTA,washoncew/completemedium,andcountcellnumber.SUSPendcellsinEPBSat~1x107/ml(Typicalyieldis~2x107cells/flask)Add800µlofcellstocuvetteplusDNA(dissolvedin10µlofsterilewater)TIPS:Youcan"tuseDNAdissolvedinTEbecauseTriswillkillcellsafterelectroporation.Onice,15minElectroporationusingGenePulser(Setvoltage:370V,Capacitance:960µF)
   1. wipeoffsolutionoutsidethecuvetteplaceintotheholder
   2. presstworedbuttonssimultaneouslyuntilyouhearbeep
   TIPS:Resultingtimeconstantshouldbearound15msec.Ifelectroporationwassuccessful,youwillseefineairbubblesoverthesolutionOnice15minUsingtinypipetincludedinthecuvettepack,transfercellsinto9mlcompletemediumin15mltube.Brieflysuspendcellsandplateinto10cmdish
2. Culture24h

Day1(Mostofcellswillnotbeattached)

1. Transfermedium(togetherwithfloatingcells)intocentrifugetubeandimmediatelyaddfreshmediumtothedishTIPS:Ifyouletthedishdryup,cellwilldie.Spindownandresuspendcellsinfreshmedium,andputbacktotheoriginaldish.
2. Culture24h.

Day2(Stillyouwillseemostofthecellsfloating)

1. Collectmedium(andcells),detachanyadherentcellsfromdishwithtrypsin/EDTA,combinethem,andspindown.
2. Resuspendcellsinselectionmediumcontainingappropriateantibiotics^*.Dispenseinto96-wellplateat200µl/well.Cultureat37°C.Youdon"thavetochangeoraddmediumuntilyouharvestthesupernatantforscreening.^*Youshouldvarynumbersofcellsthatyouplateintoeachwell.Insomecases,thetransfectionefficiencyissohighandyougetmultiplecoloniesineachwellsnecessitatingyoutore-clonethecellsfromthepositivewell.Try3x104cells/well(equivalentto3plates(300wells)/transfection)asahighestdensityandgodownto1x103/well.Ifyoudodoubletransfectionusingtwodifferentvectors(asinthecaseoftwointegrinsubunits),thenumberofcolonieswillbelowandyoumightneedmorecellsineachwells.

~Day5

Cellsfromnegativecontroltransfectionwillstarttodie.

Day7-10

Almostallofcontrolcellswillbedeadbythisperiod.Youwillbeabletoseetinycoloniesinthetransfectedwells.

Day13-16

Mediumofthewellscontainingcoloniesturntoyellow.Forsecretedproteins,takethoseculturesupthathadchangedtheircolorandscreenthemfortheexpressionlevel(byELISAetc.).Formembraneproteins,detachcellsbyadding50µltrypsin/EDTA,transferto48-or24-wells,andcheckexpressionbyFACSaftergrowingthecells.Insomecases,youmayneedadditionalre-cloningbylimitingdilution.

References

1. Stanley,Mol.Cell.Biol.9,377(1989)Davis,JBC,270,369(1995)Ikemizu,PNAS,96,4289(1999)
2. CasasnovasandSpringer,JBC,270,13216(1995)

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