Creationanduseofyourinfectiousvector: Plate5x105293Tcellsin6cm2dishescontaining5mlofmedia.(Thiscanbescaledupifdesired). Thefollowingdaysetup(usepolypropylenetubesforthis;polystyrenetubesDONOTwork!). 1ugretroviralDNAencodinggeneX 1ugpackagingplasmid(suchaspCLEco,pCLampho,pUMVC,pHR’CVM8.2deltaRetc.).Ifyouareusinga3plasmidsystemthen: forlenti:1μgpackaging(pHR’8.2deltaR)ata8:1ratiowiththeenvelopeplasmid(pCMV-VSV-G). forMuLV:thepackagingplasmid(pUMVC3)ata8:1ratiowiththeenvelopeplasmid(pCVM-VSV-G)-atotalof1ug. DMEwithoutserumto94μl 6μlFugene Mixandwait15to30minutesatroomtemperature Addto293Tcellswithouttouchingthesidesofthedish(DONOTCHANGEMEDIA). IfyouareusingamphotropicvirusthenmoveimmediatelytoBL2+inasecondarycontainer,whichhasanabsorbentmaterial.(Thisdoesnotmeanacoupleofhours;itmeansImmediately!).Therestofthisprotocolisthesameforallvirus---theBL2+safetypracticesareinplaceifyouareusingamphotropicviruses. Thefollowingdaychangethemediatowhatevermediayouwishtousewheninfectingtargetcells.293Tcellsareeasilydetachedsoremembernottoputthemediadirectlyontotocellsbutrather“run”itdownthesideofthedish.Rememberthatyouwillgetthehighesttiterviruswhenyourcellsare“happy.”Plateoutyourtargetcells. Thefollowingday.Removethemediumfromthe293Tcellsandusea0.45usyringefiltertoremoveany293Tcells.DONOTusethe0.2ufilter,asitislikelytosheartheenvelopefromyourvirusmakingitnoninfectious. Note:Afterfiltering,thefiltershouldberemovedandplacedinthebiohazardbaginthehoodandthesyringerinsedwithbleachanddecontaminatedforaminimumof20minutes.Itisusefultoplace,inadvance,aplasticbeakerwithbleachinthehoodforthis. Add8to10μg/mlofpolybrene(HexADImethrinebromide)orprotaminesulfatetothevirusandaddtothetargetcells. Carryoutinfectionfor1to4hours.Removevirusandreplacewithfreshmedia. Note:Ifyouwishtodoasecondinfectionthefollowingday,itisimportanttoputfreshmediumonthecellsandnotletthevirusremainonthecellsovernight.Themediumcontainshugeamountsofenvelopebothassociatedandunassociatedwithviralparticlesthatwillbindallthecellsurfacereceptorsrequiredforvirusadsorption,causingtheirdown-regulation.Hence,ifyoudon’tchangethemediumaftertheinitialinfection,veryfewreceptorswillbeavailableforthenextroundofinfection.Inaddition,veryfewcellstoleratethepresenceofthatmuchenvelopeforextendedperiodsoftime(i.e.alotofyourcellsmaydie). Allowthecellstorecoverandbegintoexpressthevirus-encodedgenes.Thecellsusuallyrequire48hoursforthistooccur. AdddrugifyouarescoringforthepresenceofavectorthatcarriestheappropriatedrugresistanceMarker.Priortothisstepitisadvisableeachtimeyoudoaninfectiontotitratethedrugtobeusedforselectioninordertoknowpreciselyhowmuchtoadd.Inaddition,itisnecessarytobringanextraplateofuninfectedcellsthatareoftenreferredtoas“canaries.”Adddrugtobothplates.Whenyourcanariesaredead,youcanremovethedrug. TestingforHorizontalTransfer(Whenusingthe3or4plasmidsystemyouareonlyrequiredtodothiseverycoupleofmonthsonarandomlychosenvirusprep.WhenusingthetwoplasmidsystemorpackagingcelllinesyoumustdothisEVERYtimeyoumakeanewvirusstock!!) Onceyouhavecellsthatemergedfromselectionandaregrowing,youcantestforhorizontaltransfer,i.e.fortheinadvertentgenerationofreplication-competentvirus,whichmayhaveoccurredduringthecourseofyourexperiment.Pleasenotethatthevector-infectedcells,preparedasdescribedabove,mustbegrowingand“happy.”Youshouldessentiallytreatthemasiftheywere293Tcells.Whentheyare50to75%confluent,removemediumfromthesecells,filterit,addpolybrene,andinfectyournewtargetcells. Thequestionisoftenraisedconcerningwhichcellsshouldbeusedasinfectabletargetcellsinordertotestforthehorizontaltransferresultingfromtheinadvertentcreationofinfectiousretrovirus.Thebestchoiceisacelltypethatiseasilyinfectablewiththespecificviralvectoryouhappentobeusing.UsefulcellsherearethosethathavearelativelyrapidcellcycleandareknowntobesusceptIBLetoinfectionbytheretroviralvector(andthereforetotheenvelopeglycoprotein)thatyouareusing.Atpresent(6/01),wehavefoundthatC3H10T1/2cellsareespeciallyusefulhere. Wait48hoursandaddselectiondrugtothesecellsANDtoasetofcanarycells.Bothsetsofcellsshoulddieatthesametime.Ifthecanariesdiebutthecellsinfectedbytheviralvectordon’tdosowithidenticalkinetics,thenyouhavegoodevidencethathorizontaltransferresultingfromtheinadvertentgenerationofaninfectiousretrovirushasoccurred,andallcellsandmediaresultingfromthisexperimentshouldbedestroyedbytheadditionofbleachandsubsequentautoclaving. Manyhaveaskedwhattodoiftheyhaveusedgreenfluorescentprotein(GFP)insteadofaselectabledrugmarkerinthevector.Inthiscase,youproceedexactlyasoutlinedabove.48hourslaterafterinfectionoftesttargetcells,insteadofaddingdrugyouliftthecellsandthecanariesandresUSPendthemin2%formaldehydeorparaformaldehydeandrunFACStodetermineifanyofthecellsaregreen.Youshouldrunatleast10,000cellstobesure.Ifyoudon’tknowhowtorunflowspeakwithsomeonewhodoes.Also,bothformaldehydeandparaformaldehydeautofluoresceinthegreenchannel,soitisimportantthatyouhaveasetofinfectedcellscanaryhereaswell. Selection(drugconcentrationstobeusedwithCH310T1/2cells): Neomycin1mg/ml Hygromycin300μg/ml Puromycin2μg/ml Zeocin1.2mg/ml Blastocidin15μg/ml
