Infection with retroviruses相关交流-蚂蚁淘在线

Infection with retroviruses

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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.

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Infection&nbsp;with&nbsp;retroviruses

Infection with retroviruses