pTRIPZ library subcloning Manual

ThermoScientificOpenBiosystemsExpressionArrestTRIPZLentiviralshRNAmirTechnicalManualProductDescriptionTheThermoScientificOpenBiosystemsTRIPZLentiviralInducibleshRNAmirLibrarywasdevelopedincollaborationwithDr.GregHannon(CSHL)andDr.SteveElledge(Harvard).ThislibrarycombinesthedesignadvantagesofmicroRNA-adaptedshRNA(shRNAmir)withthepTRIPZlentiviralinduciblevectortocreateapowerfulRNAitriggercapableofproducingRNAiinmostcelltypes.ThevectorisengineeredtobeTet-OnandproducestightlyregulatedinductionofshRNAmirexpressioninthepresenceofdoxycycline.ImportantSafetyNotePleasefollowthesafetyguidelinesforuseandproductionofvector-basedlentivirusassetbyyourinstitution’sbiosafetycommittee.Ingeneral,theNIHOfficeofBiotechnologyBSL2orBSL2+guidelinesshouldbefollowed.PleasenotethatTRIPZvectorsarenotcompatiblewiththirdgenerationpackagingsystemssuchasViraPowerfromInvitrogen.WerecommendtheThermoScientificOpenBiosystemsTransLentiViralPackagingSystemforusewithourvectors.DesignInformationUniquemicroRNA-30BasedHairpinDesignThermoScientificOpenBiosystemsExpressionArrestshorthairpinRNAconstructsareexpressedashumanmicroRNA-30(miR30)primarytranscripts(Figure1).ThisdesignaddsaDroshaprocessingsitetothehairpinconstructandhasbeenshowntogreatlyincreaseknockdownefficiency(Boden,Puschetal.2004).Thehairpinstemconsistsof22ntofdsRNAanda19ntloopfromhumanmiR30.AddingthemiR30loopand125ntofmiR30flankingsequenceoneithersideofthehairpinresultsingreaterthan10foldincreaseinDroshaandDicerprocessingoftheexpressedhairpinswhencomparedwithconventionalshRNAdesignswithoutmicroRNA(Silva,Lietal.2005).IncreasedDroshaandDicerprocessingtranslatesintogreatersiRNA/miRNAproductionandgreaterpotencyforexpressedhairpins.UseofthemiR30designalsoallowedtheuseof'rules-based'designsfortargetsequenceselection.Onesuchruleisthedestabilizingofthe5'endoftheantisensestrandwhichresultsinstrandspecificincorporationofmiRNAsintoRISC.Figure1.ExpressionArrestshRNAareexpressedasmiR30primarytranscripts1Theproprietarydesignalgorithmtargetssequencesincodingregionsandthe3’UTRwiththeadditionalrequirementthattheycontaingreaterthan3mismatchestoanyothersequenceinthehumanormousegenomes.EachshRNAmirconstructhasbeensequenceverifiedafterbeingclonedintothevectortoensureamatchtothetargetgene.Toassureyouthehighestpossibilityofmodulatingthegeneexpressionlevel,eachgeneisrepresentedbymultipleshRNAmirconstructs,eachcoveringauniqueregionofthetargetgene.Tet-OnSystemDesignofthepTRIPZvectorThepTRIPZvectorisengineeredtobeTet-On.TheTet-OntechnologyequipsthepTRIPZvectortoprovideforinducedexpressionofashRNAmirinthepresenceofdoxycycline().TherearetwomaincomponentsonthepTRIPZvectorenablinginduction:thetetracyclineresponseelement(TRE)andthetransactivator.TheTRE,modifiedfromitsnaturalstatetoconsistofastringofoperatorsfusedtotheCMVminimalpromoter,exhibitsreducedbasalexpressionandtighterbindingtothesecondcomponent,thetransactivator.ThepTRIPZtransactivator,knownasthereversetetracyclinetransactivator3(rtTA3)bindstoandactivatesexpressionfromTREpromotersinthepresenceofdoxycycline.ThertTA3transactivatorisamodifiedversionofthewildtypeintwoways.First,unliketheoriginaltetracyclinetransactivatorthertTA3ismodifiedtobindtotheTREinthepresenceofdoxycyclineratherthaninitsabsence.Secondly,therearethreemutationswithinthetransactivatorthatincreaseitssensitivitytodoxycyclineby25-foldovertheinitialrtTAwithoutincreasingbackgroundactivity(Das,Zhouetal.2004).UseOfTurboRFPInThepTRIPZVectorAsanaddedfeatureofthepTRIPZvector,theTREdrivestheexpressionofaTurboRFPreporterinadditiontotheshRNAmir.ThisinducedexpressionofTurboRFPenablestheusertoeasilyobserveexpressionfromtheTREpromoter,allowingquickassessmentoffactorssuchas:basalexpression,viraltiter,transductionefficiency/efficacyandoveralltechnicalsuccess.Tet-OnorTet-OffConfigurationIsPossibleThepTRIPZvectorisversatileinthatitcanbeeasilyconvertedtoaTet-OffcapablevectorusingCre/loxPtechnologyorclassicalrestrictiondigest.ThertTA3isflankedbyloxPsitesallowinginvitroorinvivoexcisionofthertTA3byexposuretoCrerecombinase.ThertTA3isalsoflankedbyapairofBamHIrestrictionsitesallowingforstraightforwardcleavageandligationofthevectortoremovethertTA3.WithoutthertTA3presentonthevectoratetracyclinetransactivator(tTA)canbeaddedextraneouslytothesystemallowingittofunctionasTet-Off®;whereexpressionofshRNAmirandTurboRFParealternativelyinducedintheabsenceofdoxycycline.ThefunctionalityandversatilityofthepTRIPZvectoristhusunsurpassedinthefieldofRNAi.VectorInformationVersatileVectorDesignFeaturesofthepTRIPZinduciblelentiviralvector(Figure2-3,Table1)thatmakeitaversatiletoolforRNAistudiesinclude:r
AbilitytousethevectorineitheraTet-OnorTet-Offconfigurationr
TurboRFPandshRNAmirarepartofasingletranscriptallowingthevisualmarkingofshRNAmirexpressingcellsr
Amenabletoinvitroandinvivoapplicationsr
InducibleRNAiexpandedtoincludebothdividingandnon-dividingcelllinesr
Puromycindrugresistancemarkerforselectingstablecelllinesr
MolecularbarcodesenablemultiplexedscreeninginpoolsFigure2.pTRIPZlentiviralvector2VectorElementUtilityTRE-minCMV