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
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
Procedure
Day-1
SplitconfluentCHOLeccellsinto75cm2flasks(1/3dilution).Day0
PreparationofDNA
Electroporation
TIPS:Resultingtimeconstantshouldbearound15msec.Ifelectroporationwassuccessful,youwillseefineairbubblesoverthesolutionOnice15minUsingtinypipetincludedinthecuvettepack,transfercellsinto9mlcompletemediumin15mltube.Brieflysuspendcellsandplateinto10cmdishDay1(Mostofcellswillnotbeattached)
Day2(Stillyouwillseemostofthecellsfloating)
~Day5
Cellsfromnegativecontroltransfectionwillstarttodie.Day7-10
Almostallofcontrolcellswillbedeadbythisperiod.Youwillbeabletoseetinycoloniesinthetransfectedwells.Day13-16
Mediumofthewellscontainingcoloniesturntoyellow.Forsecretedproteins,takethoseculturesupthathadchangedtheircolorandscreenthemfortheexpressionlevel(byELISAetc.).Formembraneproteins,detachcellsbyadding50µltrypsin/EDTA,transferto48-or24-wells,andcheckexpressionbyFACSaftergrowingthecells.Insomecases,youmayneedadditionalre-cloningbylimitingdilution.References