pMD2G慢病毒载体使用说明_精品文档资料下载.pdf

pMD2G慢病毒载体使用说明_精品文档资料下载.pdf

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哺乳动物载体；

慢病毒包装载体；

双质粒包装系统；

信哺乳动物载体；

信封载体封载体高拷贝高拷贝/低拷贝低拷贝:

高拷贝高拷贝克隆方法克隆方法:

限制性内切酶，多克隆位点限制性内切酶，多克隆位点启动子启动子:

CMV载体大小载体大小:

5824BP5测序引物及序列测序引物及序列:

CMVfwd5CGCAAATGGGCGGTAGGCGTG33测序引物及序列测序引物及序列:

-载体标签载体标签:

无无载体抗性载体抗性:

Ampicillin筛选标记筛选标记:

无无克隆菌株克隆菌株:

DH5或或HB101宿主细胞（系）宿主细胞（系）:

包装细胞系如包装细胞系如293T备注备注:

慢病毒包装载体慢病毒包装载体pMD2.G是是2质粒包装系统的信封质粒质粒包装系统的信封质粒与与psPAX2一起使用，适用范围、使用方法见下文。

一起使用，适用范围、使用方法见下文。

也可以与第三代包装载体也可以与第三代包装载体pRSV-Rev、pMDLg/pRRE一同一同使用，构成使用，构成3质粒包装系统。

质粒包装系统。

稳定性稳定性:

瞬表达瞬表达组成型组成型/诱导型诱导型:

组成型组成型病毒病毒/非病毒非病毒:

非病毒非病毒pMD2.G载体质粒图谱和多克隆位点信息载体质粒图谱和多克隆位点信息：

pMD2.G载体简介：

载体简介：

第二代慢病毒包装质粒pMD2.G使用方法-慢病毒包装与转染方法Day11.Plate5x105293Tcellsin6cm2dishescontaining5mLofmedia.（Thiscanbescaledupifdesired）Day21.Setup（usepolypropylenetubesforthis;

polystyrenetubesDONOTwork!

）:

1gretroviralDNAencodinggeneX1gpackagingplasmidMixthepackagingplasmid（psPAX2）ata8:

1ratiowiththeenvelopeplasmid（pMD2.G）-atotalof1gDMEwithoutserumto94Ltotal6LFugeneMixandwait15to30minutesatroomtemperatureAddto293Tcellswithouttouchingthesidesofthedish（DONOTCHANGEMEDIA）IfyouareusingamphotropicvirusthenmoveimmediatelytoBL2+inasecondarycontainer,whichhasanabsorbentmaterial.（Thisdoesnotmeanacoupleofhours;

itmeansImmediately!

）.Therestofthisprotocolisthesameforallviruses-theBL2+safetypracticesareinplaceifyouareusingamphotropicvirusesDay31.Changethemediatowhatevermediayouwishtousewheninfectingtargetcells.293Tcellsareeasilydetachedsoremembernottoputthemediadirectlyontotocells,butrather“run”itdownthesideofthedish.Rememberthatyouwillgetthehighesttiterviruswhenyourcellsare“happy.”2.PlateoutyourtargetcellDay41.Removethemediumfromthe293Tcellsandusea0.45usyringefiltertoremoveany293Tcells.DONOTusethe0.2ufilter,asitislikelytosheartheenvelopefromyourvirusmakingitnoninfectiousNote:

Afterfiltering,thefiltershouldberemovedandplacedinthebiohazardbaginthehoodandthesyringerinsedwithbleachanddecontaminatedforaminimumof20min.Itisusefultoplaceaplasticbeakerwithbleachinthehoodinadvance2.Add8to10g/mlofpolybrene（Hexadimethrinebromide）orprotaminesulfatetothetargetcells3.Carryoutinfectionfor1to4hours.RemovevirusandreplacewithfreshmediaNote:

Ifyouwishtodoasecondinfectionthefollowingday,itisimportanttoputfreshmediaonthecellsandnotletthevirusremainonthecellsovernight.Themediacontainshugeamountsofenvelope,bothassociatedandunassociatedwithviralparticles,whichwillbindallthecellsurfacereceptorsrequiredforvirusadsorption,resultingintheirdown-regulation.Hence,ifyoudontchangethemediaaftertheinitialinfection,veryfewreceptorswillbeavailableforthenextroundofinfection.Inaddition,veryfewcellstoleratethepresenceofhighlevelsoftheVSV-Genvelopeforextendedperiodsoftime（i.e.alotofyourcellsmaydie）Day5+1.Allowthecellstorecoverandbegintoexpressthevirus-encodedgenes.Thecellsusuallyrequire48hoursforthistooccur2.Adddrugifyouarescoringforthepresenceofavectorthatcarriesadrugresistancemarker.Priortothisstepitisadvisablethatyoutitratethedrugtobeusedforselectioninordertoknowpreciselyhowmuchtoadd.Inaddition,itisnecessarytobringanextraplateofuninfectedcells（oftenreferredtoas“canaries”）whichwillfunctionasapositivecontrolinthekillassay.Adddrugtobothplates.Whenyourcanariesaredead,youcanremovethedrug.pMD2.G载体序列载体序列：

ORIGIN1GGATCCCCTGAGGGGGCCCCCATGGGCTAGAGGATCCGGCCTCGGCCTCTGCATAAATAA61AAAAAATTAGTCAGCCATGAGCTTGGCCCATTGCATACGTTGTATCCATATCATAATATG121TACATTTATATTGGCTCATGTCCAACATTACCGCCATGTTGACATTGATTATTGACTAGT181TATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTT241ACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACG301TCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGG361GTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGT421ACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATG481ACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATG541GTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTT601CCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGAC661TTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGG721TGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCAT781CCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCCCTCGAAGCTT841ACATGTGGTACCGAGCTCGGATCCTGAGAACTTCAGGGTGAGTCTATGGGACCCTTGATG901TTTTCTTTCCCCTTCTTTTCTATGGTTAAGTTCATGTCATAGGAAGGGGAGAAGTAACAG961GGTACACATATTGACCAAATCAGGGTAATTTTGCATTTGTAATTTTAAAAAATGCTTTCT1021TCTTTTAATATACTTTTTTGTTTATCTTATTTCTAATACTTT