Infection with retroviruses相关交流-蚂蚁淘在线

Infection with retroviruses

蚂蚁淘 /发布时间:1545760489 /浏览量:184

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.

================ 蚂蚁淘在线 ================

免责声明：本文仅代表作者个人观点，与本网无关。其创作性以及文中陈述文字和内容未经本站证实，对本文以及其中全部或者部分内容、文字的真实性、完整性、及时性本站不做任何保证或承诺，请读者仅作参考，并请自行核实相关内容

Infection&nbsp;with&nbsp;retroviruses

Infection with retroviruses