Material:

• PackagingCells,e.g.Phoenixcells(anadenovirusAd5-transformedhumanembryonickidneycellline293T,transfectedwithtwoMoMLVpackaginggeneconstructs:CMV-Env-PolyA,andRSV-Gag/Pol-Tyt2-PolyA.)SeetheNolanlabwebsiteforfurtherdetailsandforobtainingandMTA.Usepassagenumber<20.
• Trypsin/EDTA(Gibco)
• RetroviralplasmidDNA(usuallybasedonMMLVbasedwithLTRs,thepackagingsignalsequenceandcontainingthegeneofinterest,apromotorandaselectableMarkerlikeantibioticaresistance,GFPorCD8)andcontrolvectorwithoutgeneofinterest.Ecotropicvectorsinfectrodentcells,amphotropicorxenotropicareabletoinfecthumancells
• 2MCaCl2(5.88gin20mLH2O0.2µmfiltered,storealiquotsat-20°C)
• 2xHBS(Hepesbufferedsaline)
• 100mMchloroquine(0.516gchloroquinediphosphate,10mLH2O,0.2µmfiltered,storealiquotsat-20°C)
• DMEMwith10%FBSand1xpenicillin-streptomycin
• Polybrene1000x(4mg/mL)inPBS,stocksterilefiltered
• Lipopolysaccharide(LPS)fromSalmonellatyphimurium,1000xstock[50µg/µL]
• Bcellmedium
• PBS
• Gentamycin500x(50mg/mL)
• Puromycin200x(300µg/mL)
• HygromycinB500x(50mg/mL)
• G418=neomycin

Method:

1. DetachthePackagingCellswith1mLTrypsin(1min/37°C),resUSPendin10mLDMEMwith10%FBS,countanaliquotandseed4.5-6x106cellsper10cmplate.Allowtogrow18-24hrpriortotransfection.
2. Prepareinasterile5mLtube20µgplasmidDNA,62.5µl2MCaCl2andH2O(sterile)ad500µl.Agitateconstantlybyanautomatedpipet(airbubbles)andadddropwise500µl2xHBS.Precipitationwilloccurwithin5minatroomtemperature.
3. Removemediafromthepackagingcells,addcarefully10mLDMEMmediumcontaining25¨µMchloroquine(2.5µl100mMstock).Addprecipitatedropwise.Incubate9-10hr.Removemediumandgentlyreplacewith5mLDMEMtocollectvirusSNfor24-36hr.
4. 12hrafterPackagingCelltransfection(whensupernatantcollectionhasbeenstarted),7.5x105MEFsshouldbeseeded(atsubconfluentdensity)per10cmplate.
5. RemovethemediumfromtheMEFs.Combine5µLpolybrene(finalconcentration4mg/mL;infectionenhancer)tothevirus-containingSNofthePackagingCellplateandfilterthrougha0.45µmfilterontheMEFplate.Addanother5mlDMEMtothePackagingCellsforongoingsupernatantcollection.
6. SuperinfectthesameplateofMEFsbyrepeatingtheinfection(asdescribedinthepreviousstep)at6and18hoursaftertheinitialinfection.
7. Allowthecellstogrowandexpresstheinfectedgenesfor24hrafterthe(last)infection.GFP(greenfluorescentprotein)encodingvectorscanbeeasilyselectedbyflowcytometricsorting.Collectsortedcellsinfreshmediumsupplementedwith200µlgentamycin500x.Alternatively,antibioticselection(correspondingtothevectorencodedresistancegene)canbecarriedout(usuallyaftersplittingthecells)in1.5-2.5µg/mLpuromycinfor2daysorin100-200µg/mLhygromycinBfor5days.Individualdrugconcentrationanddurationofselectionmayvaryandshouldresultin100%killingofuninfectedcellsascontrol.