Infectionwithretroviruses Itiswellestablishedwithavianretrovirusesthatcellsaremostefficientlyinfectedjustaftertheyaretrypsinized.Trypsinizationdoestwothings.First,itapparentlyexposesthereceptortowhichthevirusbinds.Additionally,itstimulatesDNAsynthesisandcelldivision,twoprocessesthatareessentialfortheestablishmentofretroviralinfection.Additionally,itisimportanttodotheinfectioninassmallavolumeofmediumaspossIBLe.Itislikelythattheseprinciplesarealsorelevanttotheinfectionofmammaliancellswithmurineretroviruses.
Infectionwithretrovirusesisfacilitatedgreatlybypolybrene.Thisisasmall,positivelychargedmoleculethatbindstocellsurfacesandneutralizessurfacecharge.Thisapparentlyallowstheviralglycoproteinstobindmoreefficientlytotheirreceptors,becauseitreducestherepulsionbetweensialicacid-containingmolecules.Tousepolybreneoptimally,thecellstobeinfectedshouldbepre-treatedwithpolybreneandthevirusadsorptionshouldbedoneinthepresenceofpolybrene.Cellsvaryintheamountofpolybrenethattheywilltolerate.From1to10µgpermlisusual.SomeofthesevaluescanbefoundintheCellLineHyperCardstack.
Therearetwowaystoinfectadherentcells.WhatIlikeistoseedthecellstobeinfectedinmediumcontainingpolybreneandwaituntilthecellsattachtothedish.Thiscantakefrom1to4hrs.Thenremovethemedium,addthevirus,letitadsorbfor30to60minandthenaddbackmediumcontainingpolybrene.Alternatively,youcansUSPendtrypsinizedcellsinasmallvolumeofmediumcontainingthevirusandletadsorptionoccurwhilethecellsuspensionisincubatedat37°C.Theinfectedcellscanthenbeseededondishes.
RetroviralinfectionrequirescellularDNAsynthesis--becauseviralDNAsynthesisrequiresmanyofthesamecomponentsandenzymesascellularDNAsynthesis,andmitosis--becausetheviralDNAmustgainaccesstochromosomalDNAandcannotenterinterphasenuclei.Whatisusuallydoneistoinfectcellsatadensitysuchthattheywillgrowforabout3daysbeforereachingconfluence.Theplatingefficiencyofmammaliancelllinesisusuallyhigh,closeto100%.Thedoublingtimeisapproximately18to24hr.Therefore,ifyouseed1x105cellsona50mmdish,youwillprobablyhave2x10524hourslater.Confluenceisatapproximately1063T3cellsper50mmdish.
Infectinglymphoidcells.Non-adherentcellscanbepelletedandthenresuspendedinmediumcontainingaretrovirusandanappropriateconcentrationofpolybreneandincubatedwiththevirusfor30to60min.Thecellscanthenbedilutedwithmediumandallowedtogrow.Wehavehadgoodsuccessrecentlyinfectinglymphoidcellsbyco-cultivationofthemwithvirus-producingcellsfor18hoursinmediumcontainingpolybrene.InthecaseofCOScells,thetransfectedCOScellsdon"tsurvivelongenoughtocontaminatethelymphoidcells.
