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The Impact of Transfection Mediated Toxicity -

Gene Expression and Cytotoxicity Analysis of

Transfection Reagents

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TheImpactofTransfectionMediatedToxicity-

GeneExpressionandCytotoxicityAnalysisofTransfectionReagents

Introduction

WhileplasmidDNAdeliveryisawidelyusedmethodtostudycellularfunctionsofproteinsofinterest,studiestoidentifynontoxicgenedeliveryreagentsarelimited.WiththeadventofhighͲinformation

contenttechnologies,especiallyRTͲqPCRarray,itisnowpossibletoidentifythe gen

eexpressionresponsetoaparticularcellularinsult.Thisimprovement,coupledwiththeobservationthatvirtuallyall

toxicresponsesareaccompaniedbychangesingeneexpression,suggeststhatgeneexpressionanalysis hasthepotentialtorefine

theidentificationofminimalͲtoxicitytransfectionreagentwherephenotypicresponsessuchasalteredmorphologyisnotimmediatelyevident.Consequently,weconductedan

integrativestudytoexploretheconventionaltoxicologicalendpointsandtoidentifythe

minimalͲtranscriptomiceffectsofTransIT®ͲLT1,TransIT®Ͳ2020andLipofectamine®2000TransfectionReagents

usingquantitativereversetranscriptasePCR(RTͲqPCR)arrayandpathwayanalysissoftware.

Results

EffectofTransfectiononCellMorphology

andViabilityToevaluatearoleoftransfectionͲrelatedtoxicity,timecourseanddoseͲdependentexperimentswere

conducted.HelacellsweretreatedwithvariousconcentrationsofpDNA/transfectionreagent complexesforupto24hours.Transfection

toxicitywasthenevaluatedbymorphology(Figure.1)andbyLactateDehydrogenase(LDH)leakageassays(Figure.2B).Nochangeincellularmorphologywas

observedafter8hourtreatmentofthecellswithcomplexofpDNAandanytransfectionreagents(Figure.1A).HowevertreatmentofthecellswiththecomplexofpDNAandLipofectamine®2000for24

hourscausedsubstantialmorphologicalchangesassomecellswereshrunkenandmanyweredetached

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fromthebottomoftheculturedish(Figure.1B).Nosuchchangesinmorphologywereevidentinthe cellsthatweretreatedwitheitherpDNA/TransIT®ͲLT1orpDNA/TransIT®Ͳ2020complexes(Figure.1B). ThemorphologicalchangesassociatedwithLipofectamine2000treatmentisalsoaccompaniedby massivecelllossasevidencedinlowertotalRNAyieldsfromthesesamples(Figure.2A).Similarresults werealsoobtainedwiththeLDHleakageassay.AsshowninFigure2B,treatmentwiththecomplexof luciferaseencodingpDNAandLipofectamine2000atvariousdosescaused20-55%lossofviabilityin Helacells,whereasnosignificantlossofviability(0-10%)wasfoundincellsthatweretreatedwith eitherpDNA/TransITͲLT1orpDNA/TransITͲ2020complexes.Moreover,theluciferaseexpressionlevel fromthecellsthatweretransfectedwithLipofectamine2000wasalsoreducedcomparedtothecells thathavebeentransfectedwitheitherTransITͲLT1orTransITͲ2020.

Figure1.MinimalMorphologicalChangesareobservedwithTransIT®ͲLT1andTransIT®Ͳ2020Transfection

ReagentsComparedtoLipofectamine®2000at24Hours.HeLacellsweretransfectedwithaGFPencoding

plasmidDNAusingeitherTransIT®ͲLT1,TransIT®Ͳ2020orLipofectamine®2000atreagentͲtoͲDNAratiosof3:1

for allreagents.PhasecontrastandGFPimagesweretakeninthesamefieldofviewat8(A)and24(B)hours

postͲtransfection.CellstransfectedwithTransIT®ͲLT1,TransIT®Ͳ2020remainhealthyafter24hourswhile

maintaininghightransfectionefficiency,whereascellstransfectedwithLipofectamine®2000displaysignificant

cytotoxicityafter24hours.Representativedatafromtwoindependentexperimentsisshown.

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EffectofTransfectiononGeneExpression

Inresponsetoenvironmentalchanges,cellsaltertheirgeneexpressionprofile.Toinvestigatetheroleof transfectioninducedcytotoxicityongeneexpression,anonͲcodingplasmidwastransfectedintoHela cellswitheitherTransITͲLT1,TransITͲ2020,orLipofectamine2000attheratioof3:1whichisthe optimaldosedeterminedbythepreviousexperiment(Figure.2B).QuantitativeRTͲPCRͲbased measurementsofmRNAlevelswasusedtoanalyzetheexpressionof84keygenesinvolvedin cytotoxicityat8and24hourposttransfection.Theresultwasnormalizedwiththatweretreatedonly withpDNA.Alterationingeneexpressionwasassessedusinga2Ͳfoldincrease/decreasecutoffandthe probabilityvalueoflessthan0.05.Expressionof9and25transcriptswassignificantlyalteredby pDNA/Lipofectamine2000treatmentat8and24hourrespectivelywhereasfewergenes,3and9,were affectedbytreatingthecellswithpDNA/TransITͲLT1orpDNA/TransITͲ2020complexesrespectively

Figure2.TransIT®BroadSpectrumReagents

BalanceHighEfficiencyDeliverywithLowToxicity.

(A)RNAwasharvestedfromHeLacellsthatwere transfectedwithanonͲcodingplasmidand TransIT®ͲLT1,TransIT®Ͳ2020orLipofectamine®2000 at8and24hours.TotalRNAlevelsweredecreased insamplestransfectedwith

Lipofectamine®2000

for24hours.(B)HeLacellsweretransfectedwith luciferaseencodingplasmidDNAusingeither TransIT®ͲLT1,TransIT®Ͳ2020orLipofectamine®

2000for24hours.Transfectionwasmeasuredby

luciferaseactivityusingaconventionalassay.

CytotoxicitywasassessedbyquantifyingtheLDH

releasedfromthecytosolofdamaged cells comparedtocellsalone.Highertransfection efficiencyandlowercytotoxicitywasobservedin cellstransfectedwithTransIT®ͲLT1andTransIT®Ͳ

2020atoptimalratioscomparedtocellstransfected

withLipofectamine®2000.Representativedata fromtwoindependentexperimentsisshown.

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(Figure.3AͲD).ResultsarepresentedasVenndiagramdisplayingthecommonalityoftranscriptswith2Ͳ foldorgreaterchangesinexpressionbetweenthecellstransfectedwitheachreagentfor8and24hours (Figure.3CandD).Notably,mostofthetranscriptsthatweredifferentiallyregulatedbytransfectingthe cellswitheitherpDNA/TransITͲLT1orpDNA/TransITͲ2020forboth8and24hourswerealso differentiallyregulatedwithpDNA/Lipofectamine2000treatment(Figure.3CandD).

Figure3.MirusTransIT®TransfectionReagentsMinimizetheStressResponseinTransfectedHeLaCells.StressͲrelated

geneexpressionchangesweredeterminedbyRTͲqPCRfromtotalRNAsamplesharvestedfromHeLacellsthatwere

transfectedwithanonͲcodingplasmidandTransIT®ͲLT1,TransIT®Ͳ2020orLipofectamine®2000at8

(A)and24(B)hours.

EightyͲfourgeneswereanalyzedusingtheHumanStressResponse96StellARray™(Lonza).Bargraphs(AandB)andVenn

diagrams(CandD)displaythegeneexpressionalterations.Atbothtimepoints,thenumberandmagnitudeofstressͲ

relatedgeneexpressionchangeswerelowerwhen cellsweretransfectedwithTransIT®ͲLT1orTransIT®Ͳ2020thanwhen

cellsweretransfectedwithLipofectamine®2000.Representativedatafromtwoindependentexperimentsisshown.

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BiologicalPathwaysAffectedbyTransfection

Biologicalthemesunderlyingtheexpressionpatternfromthecellsthatweretransfectedwithvarious reagentsfor24hourswereidentifiedbasedontheoverrepresentationofpredefinedgroupsof transcripts(Figure.3BandD).AsshowninFigure.4A,themostoverrepresentedcanonicalpathways includepathwaysassociatedwithoxidativestressresponseandxenobioticmetabolism.Thenegativelog ofthepvaluesfortheprobabilityofobtainingthesegenesassociatedwiththegivenpathwaysby randomchanceisgivenatthetop,whereasthebarlineinthebottomgraphisderivedfromtheratioof thenumberofgenesonourlistassociatedwithagivenpathwaydividedbythetotalnumberofgenes thatmakeupthatpathway.Amongthegenesinvolvedinoxidativestressresponse,CHOPS(DDIT3) mRNAexpressionwasincreasedapproximately50Ͳfoldinthecellsthatweretransfectedwith pDNA/Lipofectamine2000,whereastheexpressionofupto15Ͳfoldwasobservedthecellsthathave beentransfectedwitheitherpDNA/TransITͲLT1orpDNA/TransITͲ2020(Figure.3AandB).Basedon pathwayanalysis,themostsignificantbiologicalfunctionaffectedinresponsetotransfectingthecells withLipofectamine2000isrelatedtounfoldproteinandDNAdamageresponse(Figure.4B).This pathwayincludesderegulatedgenescodingforheatshockproteins;heatshock70kDaprotein5 (HSPA5),heatshock70kDaprotein1(HSPA1A),DnaJhomolog,subfamilyB,member9(DNAJB9)and DNAdamageandcellcycleregulation;cyclinͲdependentkinaseinhibitor1A(CDKN1A)andtumor protein53(TP53).

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Discussion

Inthisstudy,wehaveshownthatbothTransITͲLT1andTransITͲ2020transfectionreagentsdisplay minorcytotoxicityandhaveminimaloffͲtargeteffects,whichisvitalintheinterpretationof experimentalresults.Researchersmayintroduceadditionalexperimentalbiasdependingonthe transfectionreagentthatisselected.Toxicityeffectsincludingcellmorphology,viability,and deregulationingeneexpressionprofilewasevidentwhentreatingthecellswithpDNA/Lipofectamine

2000complexesfor24hours.Remarkably,significantchangesingeneexpressionwasalsoobserved

aftertreatingthecellswithpDNA/Lipofectamine2000complexfor8hourwhilenochangeincellular morphologywasevident.ThesestudieswereperformedinHelacellsandtheyserveasamodel;itcan

Figure4.TransfectionswithTransIT®ͲLT1or

TransIT®Ͳ2020AffectFewerCorePathwaysthan

TransfectionswithLipofectamine®2000.

Canonicalpathwayenrichmentanalysisshowsthe

primarybiologicalprocessesimpactedinHeLacells thatweretransfectedwithTransIT®ͲLT1,TransIT®Ͳ

2020orLipofectamine®2000at24hours.The

negativelogof thepvaluesistheprobabilityof obtainingthesegenesassociatedwiththegiven pathwaysbyrandomchance.Thebarlineatthe bottomofeachgraphisderivedfromtheratioof thenumberofgenesonourlistassociatedwitha givenpathwaydividedbythetotalnumber of genesthatmakeupthatpathway.Pathway analysiswasperformedusingPathwayAnalysis (IngenuitySystems).Representativedatafromtwo independentexperimentsisshown.

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beinferredthatothercelltypeswilldisplayvaryingdegreesofmorphologicalandgeneexpression changes.Dataobtainedfromgeneexpressionanalysishighlightperturbationsinpreviouslyunsuspected targetpathwaysthatareeitherbeneficialordetrimentaltothecells.Ourinvestigationsillustratethe importanceofselectingatransfectionreagentthathasminimaloffͲtargeteffects,suchasTransITͲLT1 andTransITͲ2020TransfectionReagents.

Methods

TransfectionandMicroscopy

Helacellswereseededina12Ͳwelltissuecultureplateatadensityof6×10 4 cellsperwelland incubatedinMEMwithoutPyruvate,with10%FBSfor24hours,yielding70to80%confluencypriorto transfection.CellsweretransfectedwithGFPencodingplasmid(pEGFP)usingeitherTransITͲLT1(Mirus Bio),TransITͲ2020(MirusBio),orLipofectamine2000(LifeTechnologies)transfectionreagentas recommendedbymanufacturers'protocols.Briefly,1ʅgofplasmidDNAwasdilutedinOPTIͲMEMtoa totalvolumeof100ʅl.Thesolutionwasmixedthoroughly,and1.5to5ʅloftransfectionreagentwas addedtotheDNAsolution.Aftermixing,sampleswereincubatedfor15minatroom temperatureto allowtransfectioncomplexformation.Thetransfectioncomplexeswerethenaddedtothecellsand incubatedfor24hours. GFPexpressionwasvisualizedusingsimilarexposuretimeat8and24houraftertransfectionwithan invertedfluorescencemicroscope(Axiovert200M,CarlZeiss)witha20Xairobjectiveandan appropriatefilterforGFP.Cellmorphologywasalsoexaminedunderphasecontrastoptics.Imageswere processedandanalyzedusingImageJ(NIH).

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LDHCytotoxicityAssays

Cytotoxicitywasassessedbymeasuringlactatedehydrogenase(LDH)activityreleasedfromthecytosol ofdamagedcells.Helacellswereseededina96Ͳwelltissuecultureplateatadensityof6×10 3 cellsper wellandincubatedinMEMwithoutPyruvate,with10%FBSfor24hours.Cellswerethentransfected withthecomplexesofLuciferaseencodingpDNA(pCIluc)andtransfectionreagents;TransITͲLT1(Mirus Bio),TransITͲ2020(MirusBio),andLipofectamine2000(LifeTechnologies)accordingtomanufacturers' protocolsfor16Ͳ24hours.Attheendoftreatment,themediawascollectedtomeasureLDHactivity usinganLDHͲcytotoxicitydetectionkit(RocheDiagnostics)accordingtomanufacturer'sinstructions.

TotalRNAIsolation

TotalRNAwasisolatedfromcontrolandtreatedHelacellsusingRNeasyMinikits(Qiagen)in accordancewiththemanufacturer'sinstructions.ResultingtotalRNAwasquantifiedusingNanoDrop

NDͲ1000(NanoDropTechnologies,Inc.).

cDNAPreparationandRealͲTimeRTͲPCRMeasurementofGeneExpression ComplementaryDNA(cDNA)fromeachsamplewaspreparedfrom2ugofDNaseItreatedtotalRNAby reversetranscriptionwithrandomprimers(HighCapacitycDNAReverseTranscriptionKit,Life Technologies).cDNAsampleswereanalyzedwitha96ͲpanelHumanStressResponseStellARray™qPCR Array(Lonza)onaBioͲRadrealͲtimeRTͲPCRSystem(BioͲRad)usingSYBRGreenPCRMasterMix(Life Technologies).DatawereanalyzedusingtheGlobalPatternRecognition(GPR)program(Lonza).All quantitativedataforcomparinggeneexpressionbetweendifferentsampleswereexpressedasmean. DifferencesbetweengroupswereevaluatedbytheStudent'stͲtest.Probabilityvaluesoflessthan0.05 wereconsideredstatisticallysignificant.

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BiologicalPathwayAnalysis

ListsofdifferentiallyexpressedgenesweresubjectedtoasubsequentpostͲanalysisinterrogationtofind themainbiologicalprocessesassociatedwiththeexperimentaldesign.IngenuityPathwayAnalysis (IngenuitySystems)wasappliedtoelucidatepathwaysassociatedwithtransfectionͲrelatedgene expressionchanges.ThedatasetscontaininggeneidentifiersandcorrespondingfoldͲchangeswere uploadedintothewebͲdeliveredapplication.Canonicalpathwayanalysisidentifiedpathwaysfromthe

IPAlibrarythatweremostsignificanttothedatasets.

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