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Table of Contents

Package Contents and Storage Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Storageconditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Relatedproducts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Additionalmaterialsrecommended . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Noticetopurchaser. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4RNAi Collection Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4Vector Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6FeaturesforpRSvector. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 pRSvectormap . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7FeaturesforpGFP-V-RSvector. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7pGFP-V-RSvectormap. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8FeaturesforpRFP-C-RSvector. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8pRFP-C-RSvectormap. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 shRNAinsertdescription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9Product Application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Introductionofgene-specificshRNAintomammaliancellsviatransfection . . . . 10Introductionofgene-specificshRNAintomammaliancellsviaretroviralinfection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 Stableretroviraltransduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 2 DeterminingshRNAfunctionsthroughimmunoblotting. . . . . . . . . . . . . . . . . . . . . . . . 1 2 Proteinblotting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 Proteindetectionwithspecificantibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13RNAiconstructvalidation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14PlasmidDNAamplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14Quality Control and Quality Assurance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 Plasmidvalidation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 Sequencevalidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 Transformationvalidation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15FAQ: pRS Mammalian shRNA Expression Plasmids . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

Revision10.11TS

HuSHTM

shRNA Plasmids (29-mer)

ApplicATion Guide

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pAckAGe conTenTS And STorAGe condiTionSpGFP-V-RS vectorMaterials Format Quantity

Gene-specificshRNAexpressionpGFP-V-RSvectors

Purifiedandsequence-verifiedexpressionplasmidswithgene-specificshRNAcassettes

5ugplasmidDNApervial.Fouruniquecon-structspergene.

HuSHshRNAtGFPClon-ingVector(pGFP-V-RS)(TR30007)

HuSH29-merNon-Effec-tiveScrambledpGFP-V-RS(TR30013)

Apurifiedandsequence-verifiedplasmidwithoutshRNAcassetteinsert

Apurifiedandsequence-verifiedplasmidcontain-ingnon-effective29-merscrambledshRNAcassette

5ugplasmidDNA

5ugplasmidDNA

pRFP-C-RS vector

Materials Format Quantity

Gene-specificshRNAexpressionpRFP-C-RSvectors

Purifiedandsequence-verifiedexpressionplasmidswithgene-specificshRNAcassettes

5ugplasmidDNApervial.Fouruniquecon-structspergene.

pRFP-C-RSvector(TR30014)

HuSH29merNon-Effec-tive(Scrambled)pRFP-C-RS(TR30015)

Apurifiedandsequence-verifiedplasmidwithoutshRNAcassetteinsert

Apurifiedandsequence-verifiedplasmidcontain-inganon-effective29-merscrambledshRNAcassette

5ugplasmidDNA

5ugplasmidDNA

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pRS vectorMaterials Format Quantity

Gene-specificshRNAexpressionpRSvectors

Purifiedandsequence-verifiedexpressionplasmidswithgene-specificshRNAcassettes

5ugplasmidDNApervial.Fouruniquecon-structspergene.

pRSVector(TR20003)

HuSH29-merNon-Ef-fective(Scrambled)pRS(TR30012)

Apurifiedandsequence-verifiedplasmidwithoutshRNAcassetteinsert

Apurifiedandsequenceverifiedplasmidcontaininga29-merscrambledcassette

5ugplasmidDNA

5ugplasmidDNA

Storage conditionsThedriedplasmidscanbestoredat4oC.However,oncereconstitutedwithdH

2O,

theplasmidsmustbestoredat-20oC.

Related productsPositive controls:PositivecontrolshRNAexpressionvectorsareavailableforGreenFluorescentProtein(GFP),RedFluorescentProtein(RFP)andLuciferase(Luc)inthepRS,pGFP-V-RS,andpRFP-C-RSvector.TheyhavebeenvalidatedforinhibitingtheexpressionoftheirrespectivetargetgenesintransienttransfectionexperimentsinHEK293cellswithco-transfectedGFP,RFPorLuciferaseplasmids,respectively.

TR30001[HuSH29meragainstEnhancedGFP(inpRSvector)]TR30002[HuSH29meragainstLuciferaseProtein(inpRSvector)]TR30009[HuSH29meragainsttGFP(inpRSvector)]TR30016[HuSH29meragainsttGFP(inpRFP-C-RSvector)]TR30017[HuSH29meragainsttRFP(inpGFP-V-RSvector)]

PowerPrep® HP Plasmid Purification Kits: OriGenenowoffersitsownstate-of-the-artplasmidpurificationtechnologytoitscustomersintheformofPowerPrep®HPMidiandMaxikits.Thesametransfection-gradeplasmidDNAavailableforourgene-specificproductscannowbeachievedwithPowerPrep®kits.Ourplasmidpurifica-tiontechnologyoffershigheryieldandlowerendotoxinlevelsthanmarketleadersandutilizesion-exchangecolumnseliminatingtheuseofsyringes.MoredetailedinformationonthePowerPrep®HPkitsisavailableatwww.origene.com/other/Plas-mid_Purification.

RNA validation vector (TR30004):Aconvenientproducttomeasuretheeffective-

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nessofHuSH-29orotherRNAiconstructs.ThevectorisdesignedtoincorporateacDNAcloneandaluciferasereportergeneasachimerictranscriptandcanbeusedtoidentifythemosteffectiveknockdownconstructaswellasoptimaltransfectionconditions.Highthroughputapplicationofthisreportervectorcanbeusedtoopti-mizeexperimentsinvolvingmultiplegenesandcelllines.Moreinformationcanbefoundathttp://www.origene.com/rna/validation_vector.mspx.

TrueORF cDNA expression clones:OriGenehasoneofthelargestcollectionsofcDNAclonesavailableintheworld.TrueORFcloneshavebuilt-inC-terminaltagsforeasydetectionwithanti-tagantibodies.MoredetailedinformationontheOriGeneTrueORFcollectioncanbefoundatwww.origene.com/ORF.

Additional materials recommendedTransfectionreagent:Transfectionreagentsmustbeselectedandoptimizedbasedonthecelltypebeingused.Forcellsthatareinherentlydifficulttotransfect,aret-roviralgenedeliverysystemcanbeused.OriGenesuggeststransfectionreagentslikeMegaTran1.0(OriGene),TurboFectin8.0(OriGene)orFuGENE6(Roche),whichhavebeenshowntotransfectcommoncelltypes.Celllineandcellculturesupplies:userpreferredReagentsforcelllysis:userpreferredLB-kan(25ug/ml)LB-amp(100ug/ml)LB_Chloramphenicol(34ug/ml)liquidcultureLB-kan(25ug/ml)orLB-amp(100ug/ml)LB_Chloramphenicol(34ug/ml)agarplatesReagentsandsuppliesforimmunoblots:userpreferred.OriGenehasaselectionofantibodiesanddetectionreagentsthatareavailableatwww.origene.com/anti-body/.

Notice to purchaser Thisproductisforresearchuseonly.Useinand/orfordiagnosticsandtherapeuticsisstrictlyprohibited.Byopeningandusingtheproduct,thepurchaseragreestothefollowing:Theplasmidsmaynotbedistributed,resold,modifiedforresaleorusedtomanufacturecommercialproductswithoutpriorwrittenapprovalfromOriGeneTechnologies,Inc.Ifyoudonotagreetotheaboveconditions,pleasereturntheUNOPENEDproducttoOriGeneTechnologies,Inc.withinten(10)daysofreceiptforafullrefund.

rnAi collecTion overviewAsacellulardefensemechanism,hostcellsprocessdouble-strandedRNAintosmallmoleculeswhichtargethomologousRNAsfordestruction(Hannon2002).Inmammaliancells,RNAinterference(RNAi)canbetriggeredbysiRNAsthatcausestrong,yettransientinhibitionofgeneexpressiononspecificgenes(Elbashir2001).ThesesiRNAscanbesynthesizedandtransfectedintomammaliancells,resultingineffectivesuppressionofgeneexpression.Unfortunately,suchsuppressionistran-

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sient.Bycontrast,shorthairpinRNAs(shRNA)cansuppressgeneexpressionoveraprolongedperiodbycontinuallyexpressinganRNAduplex(Brummelkamp2002;Paddison2002).

OriGenehascreatedaretroviralsilencingplasmid(pRS)thatcontainsretrovirallongterminalrepeats(LTR)fromthemurinemoloneyleukemiavirus,thepuro-mycinresistancegene,andaU6smallnuclearRNAgenepromoter(Hannon2002;Elbashir2002)toeffectivelyexpresstheinsertedhairpinsequenceandtoachieveRNAinterferenceuponintroductionintoandsubsequentprocessingbymammaliancells.ThisplasmidhasbeenvalidatedfortransienttransfectionandfortheabilitytoinhibittargetedgeneexpressionwithGFP,luciferaseandHER2oncogenespecifichairpinDNAinserts.Moreover,ourvectorhasbeenvalidatedfordownregulationofoverexpressedGFPusingretroviralinfection.Four(4)designedandsequence-veri-fiedshRNAvectorsareofferedforeachtargetedgene.

Thegene-specificshRNAexpressionplasmidswereconstructedusingsyntheticoli-gonucleotidesclonedintotheBamHI/HindIIIcloningsitesofthepRSvector.EachoftheshRNAexpressionplasmidshasa29nucleotidegene-specificsequenceinsertimmediatelydownstreamofaU6promoterinplus(+)orientation,a7nucleotideloop,andthe29nucleotidesequenceinreversecomplement,followedbyaTTTTTTterminationsequence.Allinsertshavethesequencestructureshownbelow:

U6promoter–GATCG--29ntsense–TCAAGAG–29ntreversecomplement--TTTTTT(termination)-GAAGCT

InsertionoftheshRNAcassetteintothepRSvectordestroystheBamHIrestrictionsitebychangingthesequencefromGGATCCtoGGATCG.

Approximately40shRNAvectorsweretestedagainsttheirtargetgenesinco-transfectionexperimentswheretheshRNAexpressionplasmidsareintroducedintoHEK293cellswiththeircorrespondingcDNAexpressionplasmids.Twenty-fivetothirtypercentoftheshRNAexpressionvectorswereabletoinhibittargetgeneexpressionbyatleast70%.ByprovidingfourshRNAconstructstargetingagivengene,weexpectthatatleastoneofthefourconstructsshouldprovide>90%geneexpressioninhibition.

TheOriGeneHuSHproductlinecontainsshRNAexpressionvectorscoveringmostknowngenes,suchasproteinkinases,phosphatases,oncogenes,tumorsuppres-sorgenesandothersignalingmoleculesorstructuralproteins.Foranygivengene,four(4)independentshRNAexpressionvectorsareprovidedas5ugofpurifiedanddriedplasmidpertube.Theoriginalvectorplasmid(pRSvector,TR20003;pGFP-V-RSvectorTR30007;orpRFP-C-RSTR30014)isalsoincludedinthesamepackage.Additionally,OriGeneprovidescustomerswithapRS,pGFP-V-RSorpRFP-C-RSvectorcontaininganon-effective(scrambled)shRNAcassette(TR30012,TR30013orTR30015)asaspecificnegativecontrolforgenedownregulation.Alltheexpres-sionshRNAcassettesaresequence-verified.ManyoftheHuSHproductshavebeen

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designedandannotatedtohavegoodhomologytomousesequences.Althoughourdatasuggestthatthetestedvectorswillbeabletosuppressthecorrespondinggeneexpressionby70%ormore,wehavenotvalidatedtheeffectivenessofallofferedshRNAconstructs.

PositivecontrolshRNAexpressionvectorsagainstGreenFluorescentProtein(GFP),RedFluorescentProtein(RFP)andLuciferase(Luc)genesareavailableforpurchase[pleaserefertopage3forcompletelist].shRNA-GFPandshRNA-Lucwerecon-structedinthepRSandpRFP-C-RSvectorsusingthesameapproach.shRNA-RFPwasconstructedinpGFP-V-RSvector.Eachwasshowntoinhibititsco-transfectedtargetgenebyapproximately90%whenassayedafterco-transfectionintoHEK293cellswiththeexpressionplasmidsforGFP,RFPorLuc.Aspartofthequalitycontrolprocess,eachplasmidwastransformedinto E. Coli andDNAwasisolatedfromasinglebacterialclone.DNAsequenceanalysiswasperformedoneachplasmidandthesequenceswerematchedtothespecificregionsofthetargetgenesthroughBLASTanalysis.MoreinformationisavailableontheOriGenewebsitehttp://www.origene.com/rnai/quality.mspx.

vecTor inforMATionThepRSshRNAexpressionvectorhasanumberoffeaturesallowingbothtransientandstabletransfection,aswellasthestabledeliveryoftheshRNAexpressioncassetteintohostcellsviaareplication-deficientretrovirus.EfficacyoftheshRNAexpressionvectorsshouldbedeterminedintransienttransfectionexperimentsagainstthetargetgenes.OncethesuppressingfunctionofanshRNAvectorises-tablished,thatvectorcanbeusedtocreatestablecelllines,eitherthroughtransfec-tionorretroviralinfection,viapuromycinselection.

TheOriGenepRSplasmidcontainsboth5’and3’LTRsofMoloneymurineleukemiavirus(MMLV)thatflankthepuromycinmarkerandtheU6-shRNAexpressioncas-sette.Upontransfectionoftheplasmidsintoapackagingcellline,replication-defi-cientvirusescanbeobtainedandusedtoinfecttargetcells.Apuromycin-N-acetyltransferasegeneislocateddownstreamoftheSV40earlypromoter,resultinginresistancetotheantibioticpuromycin.TheshRNAexpressioncassetteconsistsofa29nttarget-gene-specificsequence,a7ntloop,andanother29ntreversecomple-mentarysequence,allunderthecontrolofthehumanU6promoter.Aterminationsequence(TTTTTT)islocatedimmediatelydownstreamofthesecond29ntreversecomplementarysequencetoterminatethetranscriptionbyRNAPolIII.The29ntgene-specificsequencewassequence-verifiedtoensureitsmatchtothetargetgene.Adetailedmapoftheplasmidisshownonthefollowingpageandthecom-pleteDNAsequenceoftheplasmidwithoutanshRNAexpressioncassettecanbefoundathttp://www.origene.com/rna/vector_information.mspx.

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Features for pRS vector:

Start End Description1 6 EcoRI75 331 U6promoter335 340 BamHI379 385 HindIII386 391 SalI413 681 SV40promoter7431342 Puromycin-N-acetyltransferasesequence14412034 3’LTR23913010 pBR322ORI31724032 Beta-lactamaseforampicillinresistance41684639 5’LTR

pRS vector map

5’ LTR pRS shRNA Vector

5430 bp

*RC: reverse component

pBS Ori

Target Sequence Loop

29 nt

3’ LTR

U6 promoter

AMPr

SV40 early promoter

29 nt

Target Sequence RC*NNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNN TTTTTTG GAAT

GC

AAAG

Puror

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Features for pGFP-V-RS vector:InadditiontoalltheusefulfeaturesofpRS,thepGFP-V-RSvectorcontainsthepCMVdriventGFPgenewhichexpressestGFPproteinconstitutivelyinmammaliancells.Thisfeaturemakesitpossibletomonitorthetransfectionefficiency.Thebacterialselectionmarkeriskanamycin(25ug/ml)insteadofampicillin(100ug/ml)asfoundinthepRSvector.ThedetailedvectorinformationcanbefoundontheOriGenewebsite(sameURLasabove).Start End Description1 6 EcoRI75 331 U6promoter335 340 BamHI379 385 HindIII386 391 SalI413 604 SV40promoter671 1270 Puromycin-N-acetyltransferasesequence1349 1942 3’LTR2299 2918 pBR322ORI2977 3563 PolyAsignal3604 3667 MCS(PmeI(3604),NotI(3619,3649)MluI(3630),AsisI(3667)3648 4380 tGFP4467 5192 CMVpromoter5205 6095 NeomycinphosphtransferaseforKanamycinresistance6231 6701 5’LTR

Kanr pGFP-V-RS shRNA Vector

7584 bp

*RC: reverse component

pBS Ori

Target Sequence Loop

29 nt

3’ LTR

U6 promoter

CMV

SV40 early promoter

29 nt

Target Sequence RC*NNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNN TTTTTTG GAAT

GC

AAAG

Puror5’ LTR

tGFP

MCS

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Features for pRFP-C-RS Vector:ThepRFP-C-RSvectoralsocontainspCMVdriventRFPgenewhichexpressestRFPproteinconstituitivelyinmammaliancells.pRFP-C-RSalsoassiststheuserinmonitoringadual-geneknockdownefficiencywhenanshRNAcassettetargetinggeneA(expressedinRFPvector)iscotransfectedalongwithanshRNAcassettetargetinggeneB(expressedinGFPvector)alongwithrespectivetargettran-scripts.ThebacterialselectablemarkerforthevectorisChloramphenicolinsteadofAmpicillinorKanamycin.ThedetailedvectorinformationcanbefoundontheOriGenewebsite(sameURLasabove).Start End Description1 6 EcoRI75 331 U6promoter335 340 BamHI379 385 HindIII386 391 SalI413 604 SV40promoter671 1270 Puromycin-N-acetyltransferasesequence1349 1942 3’LTR2299 2918 pBR322ORI2977 3563 PolyAsignal3604 3667 MCS[PmeI(3604),NotI(3619,3649),MluI(3630)AsisI(3667)]3648 4380 tRFP44675192 CMVPromoter5263 5922CAMrforChloramphenicolresistance6104 6574 5’LTR

Camr pRFP-C-RS shRNA Vector

7452 bp

*RC: reverse component

pBS Ori

Target Sequence Loop

29 nt

3’ LTR

U6 promoter

CMV

SV40 early promoter

29 nt

Target Sequence RC*NNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNN TTTTTTG GAAT

GC

AAAG

Puror5’ LTR

tRFP

MCS

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shRNA insert descriptionTheHuSHshRNAgene-specificexpressioncassettesarepreparedusingsyntheticoligonucleotides.Theseoligonucleotidesequenceswerecomputerdesignedforoptimalsuppressionofgeneexpressionandminimaloff-targeteffects.AllshRNAse-quencesareverifiedthroughDNAsequencinganalysis.ThesequencesareprovidedtotheuserontheOriGenewebsitepriortopurchase.

TheHuSHshRNAgene-specificexpressioncassetteswereoptimizedtoincludeboththeterminationsignalforRNAPolIIIandGCcontenttargetedat50%tofurtherimprovethequalityofthegene-specificshRNAexpressionvectors.

producT ApplicATionIntroduction of gene-specific shRNA into mammalian cells via transfection

Add50uLofdH2OintoeachofthetubescontainingshRNAexpressionplasmids.

VortexthetubesbrieflytoresuspendtheDNA.Theconcentrationofthissolutionis100ng/uL.Platetheappropriateeasilytransfectedcellline(e.g.HEK293forhuman,NIH3T3formouseorOLN-93forratshRNAvalidation)cellsat3X105in2mlintoawellofa6-wellplate.Growthecellsovernightina5%CO

2incubatortoachieve50%

confluence.Inasmallsteriletube,combinethefollowingreagentsintheprescribedorder.Theorderofreagentadditionisimportanttoachievetheoptimalresults.Serum-freeDMEM 100uL TurboFection8.solution 3uLshRNAexpressionplasmidDNA 1ugcDNAexpressionplasmidforthetargetgene 0.01ugto1.0ug(optional,availableatOriGene)

Note: Add TurboFection 8.0 (or equivalent) directly into the serum-free media. DO NOT let transfection reagent touch any plastic other than the pipette tip.

For Dual-gene knockdown experiment, add 50ng of each shRNA expression plas-

mids (both pGFP-V-RS vector and pRFP-C-RS vector together) with 50ng each of target cDNAs

Mixthetubecontentsgently.DoNOTvortex!Incubateatroomtemperaturefor15-45minutes.AddtheDNA-TurboFectin8.0mixtothe6-wellplatedirectlywithoutremovaloftheculturemedia.Mixbygentlyswirlingtheplate.Incubatethecellsina5%CO

2incubatorfor48hrs.beforeharvestingforRNA

analysisand72hrs.beforeharvestingforproteinanalysis.

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Creation of stable cell lines without retroviral infectionTransfectthecellswiththeHuSHplasmidDNAusingyourstandardprotocolfortransienttransfection.Aftertransfection,donotchangethemediumuntilthecellsarereadytobepassaged.Passagethetransfectedcellsintoafreshvesselcontaininggrowthmediumand0.5-1.0ug/mlpuromycin.Continuetogrowandpassagethecellsasneces-sary,maintainingselectionpressurebykeeping0.5-1.0ug/mLpuromycininthegrowthmedium.After1-2weeks,alargenumberofthecellswillbekilledbytheantibiotic,indicatingthattheydidnottakeuporhavelosttheplasmidwiththepuromycinresistancecassette.Thecellsthatremaingrowinginthepuromycin-containingmediumhaveretainedtheHuSHplasmid,whichstablyintegratesintothegenomeofthetargetedcells.Selectclonalpopulationsofcellsbytransferringawell-isolatedsingleclumpofcells(theclonalancestorandcellsdividedfromit)intoawellofa24-wellplate;repeattoselect5-10clonalpopulations.Continuegrowingthesecellsinselec-tionmediumfor1-2additionalpassages.Atthistime,eachwellcontainsaclonalpopulationofstablytransfectedcells,whichcanbemaintainedinnormalgrowthmediumwithouttheselectionpressureofpuromycin(althoughyoumaywishtogrowthecellsunder“lightpressure”,0.2ug/mLpuromycin).Thesepopulationscanbeusedforexperimentsorstoredunderliquidnitrogeningrowthmediumwith10%DMSOand20%FBSforfutureuse.

Introduction of gene-specific shRNA into mammalian cells via retroviral infection

Production of retrovirus by transient transfection of packaging cellsPlatethepackagingcellsat40%confluencythedaybeforetransfection.Cellsshouldreach70-80%confluencyin24hours.Transfectthepackagingcellsasdescribedaboveinthetransienttransfectionprotocol.Thenextday,feedthecellculturewithfreshgrowthmedia.Onday2post-transfection,collectthemediafromtheculture,andcentrifugeat2000xgfor5minutesorpassthrougha0.45umfilter(uselowproteinbindingfilter,e.g.,celluloseacetateorpolysulfonicfilter,notanitrocellulosefilter)tore-movecelldebris.Freezethesupernatantat-800CordirectlyuseitasviralstockforviraltiteringthroughinfectionofNIH3T3cells.pGFP-V-RSvectorconveystGFPexpressionandpRFP-C-RSvectorconveystRFPexpressionupontransfection.HoweverthetGFPandtRFPexpressioncassettesarelocatedoutsideoftheretroviralpackagingregion.ThevirusdoesnotpassthetGFPortRFPintothetransducedcell.

Production of retrovirus by stable transfection of packaging cellsPlatethepackagingcellsat40%confluencythedaybeforetransfection.Cellsshouldreach70-80%confluencyin24hours.Transfectthepackagingcellsasdescribedaboveinthetransienttransfection

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protocol.At24-36hourspost-transfection,replatethetransfectedcellsinselectionmedia(completegrowthmediumwiththeselectionagentaddedatitsoptimalconcentration[e.g.,puromycinataconcentrationof0.5to1ug/mLforHEK293Tcells]).Culturethecellsforoneweekusingthedrugselectionmedium.Manyofthecellswilldieduetonegativeselection,leavingonlydrug-resistantcellsalive.Select10-20large,healthy-lookingdrug-resistantcoloniesandtransfereachintoawellofanew6-wellplate.Expandthesecoloniesintolargecultures,andcomparetheirviralyieldbyvirustitering,ifdesired.Choosethecellswiththehighesttitertouseforvirusproduc-tion.Otherwise,collectthemediafromtheculture,andcentrifugeat2000xgfor5minutesorpassthrougha0.45umfilter(uselowproteinbindingfilter,e.g.,celluloseacetateorpolysulfonicfilter,notanitrocellulosefilter)toremovecelldebris.Freezethesupernatantat-800CordirectlyuseitasviralstockforviraltiteringthroughinfectionofNIH3T3cells.

Stable retroviral transduction Platethetargetcellsataconcentrationthatwillproduceapproximately50%confluencyin24hours.Addentireamountofviralstock(or,ifvirushasbeentitered,thechosencfu/mLofvirus)and4ug/mlpolybreneingrowthmediumdirectlyontotargetcells.Incu-bateat37ºCin5%CO

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At24hourspost-infection,replacethemediumwithfreshgrowthmediumcontaining0.5-1ug/mlpuromycin(ortheoptimalconcentrationasdeterminedforyourconditions).Passageasneeded,andmaintainselectionpressurefor1-2weeks.Mostuninfectedcellsshouldbekilledbythepuromycinwithin1week.Selectclonalpopulationsofcellsbytransferringawell-isolatedsingleclumpofcells(theclonalancestorandcellsdividedfromit)intoawellofa24-wellplate;repeattoselect5-10clonalpopulations.Continuegrowingthecellsinselec-tionmediumfor1-2additionalpassages.Atthistime,eachwellcontainsaclonalpopulationofstablytransfectedcells,whichcanbemaintainedinnormalgrowthmediumwithouttheselectionpressureofpuromycin.Thesepopulationscanbeusedforexperimentsorstoredinliquidnitrogentankingrowthmediumwith10%DMSOand20%FBSforfutureuse.

Determining shRNA functions through immunoblotting

Preparing cell lysatesRemovetheculturemediabyaspiration.Washthecellsinthedishoncewithice-coldPBSandaspirateoffPBS.Addice-coldRIPA*withfreshlyaddedproteaseinhibitorstocells(1mlper10cmdish;0.2mlperwell/six-wellplate).Foradherentcells,rockthecellsinthepresenceoflysisbufferinplatesinacoldroomoronicefor15minutes.Forsuspensioncells,pelletthecells,thenresuspendinlysisbuffer.Transferthecelllysissolutionintoeppendorftubes.

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Centrifugethelysateat14,000xginapre-cooledcentrifugefor15minutes.Immediatelytransferthesupernatanttoafreshcentrifugetubeanddiscardthepellet.Determinetheproteinconcentrationbyanycommerciallyavailablereagentorkit.Atthisstep,thesamplecanbedividedintoaliquotsandstoredat–80oCforlong-term.

*RIPAbufferStock:50mMTris-HClpH7.4,1%NP-40;0.25%Na-deoxycholate1,150mMNaCl,1mMEDTAAdd fresh:1mMPMSF2,1ug/mlAprotinin,1ug/mlLeupeptin

1DonotaddNa-deoxycholatewhenpreparinglysateforkinaseassays,asitmaydenaturetheproteinand

causeittoloseactivity.2PMSFismadeasa200mMstocksolutioninisopropanolandstoredatroomtemperature.Thevaporis

hazardous.Itisimportanttoworkwithitinachemicalhood.PMSFisnotstableinH2

Oasithasahalf-life

ofapproximately30minutes.

Protein blottingPrepare3ugofcelllysatein1XLaemmlisamplebuffer1inavolumeof20uL(foramini-gel,upto15ugofproteincanbeloadedperlane).Heatthesampleto70oCfor10min.Prepareapre-stainedproteinstandardaswell.Runthesamplesonapre-castSDSpolyacrylamidegelwithTris-GlycineSDSrunningbufferat125Vfor90minutesuntilthedyereachesthebottomthegel.Removethegelandsoakinproteintransferbufferfor15minutes.PreparethePVDFmembranebypre-wettingitin100%methanol,washingonceindH

2Ofor5minandequilibratingitintheproteintransferbufferfor10minutes.

Assembletheelectroblottingcassetteandplacetheelectrodesintheblottingunit,accordingtothemanufacture’sinstructions.TransferinTris-Glycinetransferbufferat25V(100mA)for1.5hours.Followingtransfer,removethemembranefromtheblottingcassetteandmarktheorientationofthegelwithapencil.RinsebrieflywithPBSandtrimthemem-brane.Themembranemaybestoredat4oCforseveralweeks.However,oncethemembraneisdried,itneedstobewettedbymethanolfollowedbyPBS.

12XLaemmlisamplebuffer:62.5mMTris-HCl,pH6.8,2%SDS,25%glycerol,0.01%BromophenolBlue

Protein detection with specific antibodiesWashPVDFmembranewithTBST2oncefor5min.atroomtemperature.Blocknon-specificbindingonthemembranewithfreshlyprepared5%nonfatdriedmilkfor1houronashakingplatformatroomtemperature.Washthreetimesfor5minuteseachwithTBST.IncubatethemembranewithaspecificprimaryantibodydilutedinTBSTand5%BSAatthemanufacturer’srecommendeddilutionwithgentleagitationat4oCovernightorforseveralhoursatRT.Washthreetimesfor5mineachwithTBST.IncubatewithHRP-conjugatedsecondaryantibodyat1:20,000(ormanufacturer’s

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recommendeddilution)inTBST-5%BSAfor1houratroomtemperature.Washthreetimesagainfor5minuteseachwithTBST.Fordetection,usetheenhancedchemiluminescencereagentfromOriGene(West-ernBlottingLuminolReagent(TA10006))orothercommerciallyavailabledetec-tionsystemandprepareaccordingtothemanufacturer’sdirections.Laythemembraneonaplasticsurfacewiththeproteinsideup.Addthemixeddetectionsolutiontothemembrane.Incubatefor3minutes.Removetheexcesssolutionandcoverthemembranewithtransparentplastic.PlacethewrappedblotwithproteinsideupinanX-rayfilmcassette.PlaceasheetofX-rayautoradiographyfilmonthetopofthemembrane.Closethecas-settefor1min.Removethefilmfordevelopment.Addadditionalfilmsifneededforlongerorshorterexposures.

2TBST:10mMTris-HCl,pH8.0,150mMNaCl,0.05%Tween20.

RNAi construct validation (Optional)

ThemostuptodateprotocolforthisprocesscanbefoundontheOriGenewebsiteathttp://www.origene.com/assets/Documents/HuSH/ValidationVectorProtocol.pdf

Plasmid DNA amplification (Optional)For pRS based HuSH vectors, the E. coli selection marker is ampicillin (100 ug/mL). For pGFP-V-RS based HuSH vectors, the E. coli selection marker is kanamycin (25 ug/mL). For pRFP-C-RS based HuSH vectors, the E. coli selection marker is chloram-phenicol (34 ug/ml)

Ifdesired,customerscanfirstamplifytheshRNAvectors.Add50uLofdH2Ointo

eachtube.VortexthetubesbrieflytoresuspendtheDNA.Pipette1uLofthissolu-tiontoanothertubeandadd99uLdH

2O.TheconcentrationoftheDNAsolution

shouldbearound1ng/uL.Theplasmidsolutionshouldbestoredat–20oC.ThawtransformationcompetentE. Coli cells(standardlaboratoryDH5alpha)onice.Performtransformationwith1-2uLofthedilutedshRNAplasmid.PlateoutthetransformantsonLB-Kan,LB-AmporLB-Chloramphenicolplatesandincubateovernightat37oC,untilcoloniesappear.Thefollowingday,inoculatesinglebacterialcoloniesinto5mlofLB-Kan,LB-ChloramphenicolorLB-Ampmediaandgrowthemovernight.PurifytheDNAplasmidsfromthecultureusingaminiprepDNAisolationkit.ResuspendtheDNAin50uLofTEsolutionanddeterminetheconcentrationofthesamples.Storethesolutionat–20oC.

QuAliTy conTrol And QuAliTy ASSurAncePlasmid validationAllplasmidproductswithshRNAexpressioncassetteshavebeenisolatedfromsinglecolonies.Thepurifiedplasmidswereexaminedonanagarosegeltoensurethepres-enceoftheplasmidsandtoverifyquantity.

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Sequence validationThefinalproductshavebeenre-sequencedforconfirmation.

Transformation validationThedriedplasmidswereresuspendedindH

2O,thenusedfortransforming E. Coli

cells.Theefficiencyoftransformationisidenticaltothatbeforedrying.

MoreQualityControlinformationisavailableattheOriGeneweb-sitehttp://www.origene.com/rna/quality.mspx.

fAQ: prS MAMMAliAn SHrnA expreSSion plASMidSWhat are the differences between the pRS, pGFP-V-RS and pRFP-C-RS ex-pression plasmids?Answer:ThepGFP-V-RSplasmidcontainstGFPdrivenbyaCMVpromoter.ThepRFP-C-RSplasmidcontainstRFPdrivenbyaCMVpromoter.Bothplasmidsprovideaneasywaytoidentifycellsthathavebeentransfected.ThepGFP-V-RSvectoriskanamycinresistant,pRFP-C-RSvectorischloramphenicolresistantwhilethepRSvectorisampicillinresistant.

I am doing retroviral packaging and infection, will my infected cells express tGFP or tRFP?Answer:No.TheCMV-tGFPandtheCMV-tRFPelementsareoutsideoftheregionthatgetspackagedbyretrovirus.Thus,youcannotusetGFPortRFPexpressiontomonitortransductionbutyoucanusepuromycinselectiontogeneratestablecelllines.

Your HuSH product is stated to be “locus-specific”. How do I know that it will knock down the expression of my variant or isoform?Answer:Unlessstatedotherwise,shRNAconstructsweredesignedtobeeffec-tiveagainstmosttranscriptionalvariantsataparticulargenelocus.Ifyouwouldlikeaknockdownconstructagainstaspecifictranscriptionalvariant(s),OriGenecangenerateacustomHuSHproductthatwillselectivelyknockdownonlythespecifiedvariants.PleasereviewtheoptionsonourExactHuSHwebsiteatwww.origene.com/rnai/exact-HuSH.aspx

Can I screen all the constructs provided in HEK293 cells and then pick the most effective one for subsequent studies?Answer:Absolutely.Werecommendthatyouscreenallconstructsindividuallytoidentifythemosteffectiveconstructs.HEK293cellsareaconvenientandeasilytransfectablesystemforscreeningallhumanshRNAconstructs(useNIH3T3formouseshRNAconstructsorOLN-93forratshRNAconstructs).Othercelllinescanalsobeusedforthispurpose,solongasthetransfectionefficiencyofourpRS.pGFP-V-RSandpRFP-C-RSvectorishigh,~70%orgreaterfortransienttransfec-

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tions.Afterwards,theeffectiveconstruct(s)canbeusedforretroviralinfectionoforfordirecttransfectionintoyourtargetcells.Can I get a HuSH construct against any species other than those on your web site?Answer:OriGenecandoanycustomHuSHdesignandconstruction,regardlessofspeciesspecificity.Theonlyinformationyouneedtoprovideistheaccessionnumberofsequenceyouwishtotarget.Wecanofferyouassistanceinidentifyingtheparticularspecies’sequencestobeusedforgeneknockdownstudiesoryoucanfollowthelinktoourExactHuSHpagehttp://www.origene.com/rnai/exact-HuSH.aspx

Will your HuSH products work with any retroviral packaging cell line?Answer:OurpRS,pGFP-V-RSandpRFP-C-RSvectorshavebeendesignedforviralpackaginginmostcommerciallyavailablepackagingcelllines.However,pleasemakesurethatthepackaginglinehasnotbeenpreviouslytransfectedwithplas-midscontainingapuromycinresistancecassette.Furthermore,youneedtoen-surethatthechosenpackagingline’sviralparticlesareabletoinfectyourtargetcellline(somecelllineshaverestrictedspeciesspecificity).WesuggestpackaginglinessuchasPT67(Clontech)orPhoenix(Orbigen)forusewithourconstructsforcelllineinfection.

How should I use the products?Answer:Customerscanusethe5ugshRNADNAplasmiddirectlyfortransfec-tionandgene-knockdownstudies.Aftertransfection,celllysatescanbeobtainedandusedforWesternblotanalysiswithanantibodyagainstthetargetproteintoverifythefunctionalityoftheshRNAvectors,orRNAcanbeharvestedfromtransfectedcellsandusedinquantitativeRT-PCRtodeterminethelossofgeneexpression.Ifdesired,theshRNAplasmidscanbere-transformedforunlimitedsuppliesoftheplasmids.

Can I select for the plasmid-transfected cells?Answer:Yes.Onedayaftertransfection,thecellscanbeselectedwithculturemediacontaining0.5-1.0ug/mLpuromycin(Sigma).

How can I create a stable cell line with a functional shRNA expression vec-tor? Answer:Stablecelllinescanbegeneratedbytwodifferentmethods.First,targetcellscanbetransfectedwiththeshRNAplasmid.Onedayaftertransfection,thetransfectedcellscanbeselectedwith0.5-1.0ug/mlpuromycinfor1-2weekswithpassagesasneeded.Alternatively,retroviralpackagingcelllinescanbeusedtogenerateretrovirusesforstablecelllinegeneration.Forexample,theplasmidcanbetransientlytransfectedintothePhoenixhelper-freeamphotropicpackagingcellsline.Twodaysafterthetransfection,thecellculturesupernatantiscollected,filteredandusedtoinfectthetargetcelllines.Stablecelllinescanbeestablishedfollowingdrugselection.

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What use does the empty vector serve? Answer:ForanyexperimentinvolvingtheintroductionofforeignDNAintocells,itisimportanttoeliminateanynon-specificeffectsbyusinganemptyvec-tornegativecontrol.ThevectorscanalsobeusedforcloningyourownshRNAconstruct(s).

What use does the scrambled non-effective plasmid serve?Answer:Tospecificallyruleoutthepotentialnon-specificeffectinducedbyexpres-sionoftheHuSHproduct,OriGeneprovidescustomerswithanegativecontrol(TR30012,TR30013orTR30015),thatwasconstructedbycloningascrambledsequencecassette(5’GCACTACCAGAGCTAACTCAGATAGTACT3’)intoourpRS,pGFP-V-RS,orpRFP-C-RSvectorsrespectively.Theplasmidshouldserveasanega-tivecontrolforgene-specificknockdownexperimentsandexcludeanypotentialinterferonresponse.

Can you tell me the sequence of your control constructs?Answer:The29mersequenceusedtotargetfireflyluciferaseinTR30002is5’GGATTTCAGTCGATGTACACGTTCGTCAC3’.The29mersequenceusedtotargeteGFPinTR30001is5’CACAAGCTGGAGTACAACTACAACAGCCA3’,the29mersequenceusedtotargettGFPinTR30009andTR30016is5`GCTACGGCTTCTAC-CACTTCGGCACCTAC3`,andthe29mersequenceusedtotargettRFPinTR30017is5’CTTCAAGACCACATACAGATCCAAGAAAC3`.Thenon-effectivecontrolse-quenceis5’GCACTACCAGAGCTAACTCAGATAGTACT3’.

Do I need to use a special strain of E.coli to amplify my HuSH constructs? Answer:SpecialE.coliarenotrequiredforHuSHamplificationbutwedonotrec-ommendusingJM109.WeroutinelyuseDH5alphafromNewEnglandBiolabs.

Are the HuSH plasmids high copy or low copy number?Answer:Althoughtheplasmidstechnicallycontainahigh-copynumberOriofreplication,thehairpinslowsreplicationandthus,werecommendusingalow-copynumbermethodforplasmidDNAamplification.

Can the pRS, pGFP-V-RS or pRFP-C-RS vectors be packaged by lentivirus? Answer:No,ourcurrentRSsystemisstrictlyretroviral.However,weareinthepro-cessofcreatingalentiviralHuSHvector.Pleasecheckourwebsiteforitsrelease.

Do I have to use retroviral packaging and infection to create stable cell lines?Answer:Ifyourtransfectionefficiencyisveryhigh(e.g.80%orgreater),itisnotnecessarytouseretroviralpackaging.Simplysplityourcells24hrs.post-transfec-tionandaddpuromycin(0.5-1ug/ml)tothefreshgrowthmedium.

Will 0.5ug/ml-1ug/ml concentration of puromycin work for my cell line? Answer:Westronglyrecommendthatakill-curvebeperformedoneachbatchofcellstoensurethattheoptimalpuromycinconcentrationisemployed.

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What is the OriGene guarantee on the shRNA expression plasmids?Answer:OriGeneguaranteesthatthesequencesintheshRNAexpressioncas-settesareverifiedtocorrespondtothetargetgenewith100%identity.Oneofthefourconstructsatminimumareguaranteedtoproduce70%ormoregeneexpres-sionknock-downprovidedaminimumtransfectionefficiencyof80%isachieved.WesternBlotdataisrecommendedoverqPCRtoevaluatethesilencingeffectoftheshRNAconstructs72hrsposttransfection.Toproperlyassessknockdown,thegeneexpressionlevelfromtheincludedscramblecontrolvectormustbeusedincompari-sonwiththetarget-specificshRNAtransfectedsamples.

Fornon-conformingshRNA,requestsforreplacementproductmustbemadewithinninety(90)daysfromthedateofdeliveryoftheshRNAkit.Toarrangeforafreereplacementwithnewlydesignedconstructs,pleasecontactTechnicalServicesattechsupport@origene.com.Pleaseprovideyourdataindicatingthetransfectioneffi-ciencyandmeasurementofgeneexpressionknockdowncomparedtothescrambledshRNAcontrol(WesternBlotdatapreferred).

What if the gene is not expressed in HEK293 cells and the transfection effi-ciencyofmytargetcellsisbelow80%?HowdoIscreenmyshRNAconstructstopickthemosteffectiveone?Answer:IfyourgeneisnotexpressedinHEK293,youcandoaco-transfectionwithanexpressionconstructandtheshRNAconstructata1:1ratiotransientlyintoHEK293cells.Youcanalsoselectstablecellsinyourtargetcellline.Itiswellknownthatifagenethatisvitalforcellgrowthissilenced,itwillbedifficulttogetstablecellsforthatparticularcellline.

Which method do you recommend for assessing the knockdown efficiency of my gene-specific shRNA constructs?Answer:WesternBlotisrecommendedoverqPCRtoevaluatethesilencingeffectoftheshRNAconstructs72hrsposttransfection.Toproperlyassessknockdown,thegeneexpressionlevelfromtheincludedscramblecontrolvectormustbeusedincomparisonwiththetarget-specificshRNAtransfectedsamples.

referenceSBrummelkampTR,BernardsR,AgamiR.(2002)AsystemforstableexpressionofshortinterferingRNAsinmammaliancells.Science.296:550-3.ElbashirSM,HarborthJ,LendeckelW,YalcinA,WeberK,TuschlT.(2002)Du-plexesof21-nucleotideRNAsmediateRNAinterferenceinculturedmammaliancells.Nature411:494-8.HannonGJ.(2002)RNAinterference.Nature.418:244-51LuoB,HeardAD,LodishHF.(2004)SmallinterferingRNAproductionbyenzy-maticengineeringofDNA.ProcNatlAcadSciUSA.101:5494-9.PaddisonPJ,CaudyAA,BernsteinE,HannonGJ,ConklinDS.(2002)ShorthairpinRNAs(shRNAs)inducesequence-specificsilencinginmammaliancells.GenesDev.

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16:948-58.SenG,WehrmanTS,MyersJW,BlauHM.(2004)Restrictionenzyme-generatedsiRNA(REGS)vectorsandlibraries.NatGenet.36:183-9.ShiraneD,SugaoK,NamikiS,TanabeM,IinoM,HiroseK.(2004)Enzymaticpro-ductionofRNAilibrariesfromcDNAs.NatGenet.36:190-6.

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