Promoter, transgene, and cell line effects in the transfection of mammalian cells using PDMAEMA-based nano-stars

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Promoter, transgene, and cell line effects in the transfection of mammalian cells using PDMAEMA-based nano-stars

Alexander Raup

^a

, Valérie Jérôme

^a

, Ruth Freitag

^a,

*, Christopher V. Synatschke

^b,1

, Axel H.E. Müller

^b,2

aProcessBiotechnology,UniversityofBayreuth,95440Bayreuth,Germany

bMacromolecularChemistryII,UniversityofBayreuth,95440Bayreuth,Germany

ARTICLE INFO

Articlehistory:

Received7March2016

Receivedinrevisedform9May2016 Accepted9May2016

Availableonline16June2016

Keywords:

Fluorescentreporterprotein Co-expression

Non-viralgenedelivery Mammaliancell

Poly(2-dimethylamino)ethylmethacrylate

ABSTRACT

Non-viral transfectionprotocolsare typicallyoptimized usingstandardcells andreporterproteins, potentiallyunderestimatingcellularortransgeneeffects.Heresucheffectswerestudiedfortwohuman (Jurkat,HEK-293)andtworodent(CHO-K1,L929)celllinesandthreeﬂuorescentreporterproteins.

Expressionoftheenhancedgreenfluorescentprotein(EGFP)wasstudiedunderthecontrolofthehuman elongationfactor1 alphapromoterandthreeviralpromoters(SV40,SV40/enhancer,CMV),thatof ZsYellow1(yellowfluorescence)andmCherry(redfluorescence)fortheCMVpromoter.Resultsvaried withthecellline,inparticularfortheJurkatcells.Pair-wiseco-transfectionoftheCMVcontrolled transgenesresultedinasignificantfractionofmonochromaticcells(EGFPforEGFP/YFPandEGFP/RFPco- transfections,YFP incase ofYFP/RFPco-transfections). OnlyJurkat cellswerealmost incapable of expressingYFP.DilutionoftheplasmidDNAwithanon-expressedplasmidshowedcelllinedependent effectsontransfectionefficiencyand/orexpressionlevels.

ã2016TheAuthor(s).PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).

1.Introduction

Recombinantproteinproductioninmammaliancellsrequires theintroductionoftherespectiveDNAsequencesintothecells’ nuclei(‘transfection’).Forbiotechnologicalapplications,non-viral transfectionmethods,involving,e.g.,polycationssuchasPEI(poly (ethyleneimine)) or PDMAEMA (poly(2-dimethylamino) ethyl methacrylate), are preferred [1,2]. According to current under- standing, the role of the polycationis tocompact and charge- compensatetheplasmidDNA(formationof‘polyplexes’),thereby facilitatinguptake bythecells. Dependingonitschemistry,the transfectionagentmaythenalsoaidendosomalescape(e.g.viathe

‘protonsponge’effectdescribedforPEI[3]),transportthroughthe cytosol,andﬁnallyreleaseintheperinucleararea.Inspiteofsome undeniableprogressoverthelastdecade[4],non-viraltransfec- tionagentsarestillordersofmagnitudelessefﬁcientthanviral ones [5]. This has led to intensive research on the effect of

chemistryand structureofnon-viraltransfectionagents.Inthis context,itisgenerallyassumedthatthepolycationdeterminesthe transfectionefﬁciency,i.e.thepercentageofcellsthattakeupthe DNA, but transfects the DNA indiscriminately of the encoded information.Differencesintransgeneexpressionstrength,onthe otherhand, aredeterminedby featuresof thetransfected DNA such as the promoter. Promoters are known to show some variabilityindifferentcelllines.However,thecomplianceoftheir performancewithsomegeneraltrends,e.g.inregardtorelative strengthandspecies-dependency,istypicallyassumed[6–9].

Since thePDMAEMA chemistry is particularly suited tothe synthesisofwell-defined homopolymerswithvariedtopologies, muchofourknowledgeonthestructure-function-relationshipof polycationic transfection agents was derived from experiments involving PDMAEMAs [2]. From these studies it was inter alia deducedthatnon-linearpolycationsaresignificantlylesscytotoxic thanlinearonesofthesamesizeandchemistry[10].Basedonthis observation,ourgrouphasrecentlydemonstratedthatPDMAEMA- based star-shaped nanoparticles have high potential for trans- fecting mammalian cells including primary human blood cells [11,12]. Fine-tuning of the respective transfection protocols in terms of optimizing efficiency and biocompatibility typically involved the transfection bufferin terms of composition, ionic strength,andpHaswellastheN/Pratio(amountofnitrogeninthe

* Correspondingauthor.

E-mailaddress:ruth.freitag@uni-bayreuth.de(R.Freitag).

1 Presentaddress:SimpsonQuerreyInstituteforBioNanotechnology, North- westernUniversity,Chicago,IL60611,USA.

2 Presentaddress:InstituteofOrganicChemistry,Johannes-Gutenberg-Univer- sity,55099Mainz,Germany.

http://dx.doi.org/10.1016/j.btre.2016.05.003

2215-017X/ã2016TheAuthor(s).PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

ContentslistsavailableatScienceDirect

Biotechnology Reports

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polymer/amount of phosphate in pDNA) of the polyplexes.

Pronounced heterogeneities in transgene expression were ob- served among the cells of a given transfection batch in these experiments,whiletheestablishmentofatrulygenerictransfec- tionprotocolhassofarbeenelusive[13].

The basis for theinvestigation of transfectionoutcomes has beenchangedsomeyearsagobytheadventofﬂuorescentreporter proteins[14].Thesetransgenesallowadirectstatisticalevaluation ofthedistributionoftheexpressionstrengthovertheindividuals ofa(living)cellpopulationbyﬂowcytometry.Inconsequenceit becomes possible to differentiate whether a given amount of transgeneisproducedbyasmallnumberofhighproducerswithin thepopulationorbyalargenumberexpressinglowlevelsofthe protein.Incontrast,onlyaveragevaluescanbedeterminedinthe caseofreporterproteinsrequiringenzymaticconversionofadded substratesfordetection,suchasluciferaseorß-galactosidase[15], sincetheseassaysarebynecessityperformedintherespectivecell lysates.

Inviewofthewidespreaduseofrecombinantreporterproteins astools,surprisinglylittlecanbefoundintheliteratureintermsof asystematicinvestigationoftheirtransfectiontakingthevarious putativeimpactfactorsintoaccount.Anareawherethiscouldbeof particular importance is the co-transfection of a fluorescent reporterwithanother(fluorescent)transgene,whereinterference orcompetitioncouldbiastheresults.Forinstance,thecombina- tionoftwoormorefluorescentreportersisanimportanttoolin cellandtissueanalytics(imaging).Molecularbiosensorsareused tostudycellularand molecularheterogeneityor thelong-term biologicaleffectsofsignalinginstemcellresearch[16].Fluorescent proteins can also be paired for quantitative multiparameter imagingof livesystemsin vivoand invitroor forfluorescence resonanceenergytransfer(FRET)studies.Recognizedadvantages of the “two-color”-approach include the possibility of photo- switchingaswellasofbimolecularfluorescencecomplementation (BiFC) [17,18]. Since flowcytometry can be set up to quantify severalfluorescentdyesinparallel,itisasuitabletechniquefor studyingsucheffects.

Here, a popularreporter transgene, namely enhanced green ﬂuorescent protein(EGFP)under thecontrolofone outoffour differentpromoterswasinitiallytransfectedintotwohumanand two rodent cell lines to test for putative promoter effects.

Subsequently,plasmidsencodingforthisortwootherfluorescent proteins each under the control of the cytomegalovirus (CMV) immediate early promoter were transfected or (pair-wise) co- transfected into the cells. Using three different fluorescent transgenesallowedustostatisticallyquantifyspecificeffects on transfectionefficiencyaswellasonthedistributionoftransgene expressionstrengthbyflowcytometry.Toourknowledge,thisis the first time that the co-expression strength distribution of independentlytransfected reporter proteins was determined in parallel.

2.Materialsandmethods 2.1.Materials

If nototherwiseindicated,we usedPAALaboratories(Cölbe, Germany) or Greiner bio-one (Frickenhausen, Germany) as supplierforcellculturematerialsandSigma-Aldrichforchemicals.

Fetalcalfserum(FCS)wasfromBiochromAG(Berlin,Germany).

Dulbecco’s Phosphate-Buffered Saline without Ca²⁺ and Mg²⁺

(DPBS)wasfromLonza (Visp,Switzerland). HBGbuffer(20mM Hepes,5wt%glucose,pH5.5)waspreparedinhouseandsterilized byﬁltration.Cell culturemediaR10 (RPMI1640withoutgluta- mine,add10vol%fetalcalfserum,2mML-glutamine,100IU/mL Penicillin/100

m

^g/mLStreptomycin),MEM10(MEMEarle’swithout

L-glutamine/FCS, add 10vol% FCS, 4mM L-glutamine,100IU/mL Penicillin/100

m

^g/mL Streptomycin), and Opti-MEM were from Lonza(Cologne,Germany),BiochromAG(Berlin,Germany),and Thermo Fisher Scientiﬁc (Dreieich, Germany), respectively. For pre-equilibration,mediawereincubatedfor 1–4hinastandard mammaliancellcultureincubator(37C,5%CO2,95%humidity).

2.2.Cryogenictransmissionelectronmicroscopy(cryo-TEM) Forcryo-TEMstudies,adrop(2

m

^L)^of^the^aqueous^micellar

solution(concentrationca.0.5g/L)wasplacedonalaceycarbon- coated copper TEM grid (200mesh, Science Services, Munich, Germany), where most of the liquid was removed with filter paper,leavingathinfilm.Thespecimenswereshockvitrifiedby rapidimmersionintoliquidethaneinatemperature-controlled freezingunit(ZeissCryobox,CarlZeissNTSGmbH,Oberkochen, Germany) andcooled to approximately 90K. The temperature was monitored and kept constant in the chamber during the entirepreparation.Afterfreezingthespecimenwereinsertedinto acryo-transferholder(CT3500,GatanGmbH,Munich,Germany) andtransferredtoaZeissEM922OMEGAEFTEMinstrument(Carl ZeissNTS GmbH).Measurements werecarried outat approximately 90K. The electron microscope was operated at an accelerationvoltageof200kV.Zero-lossfilteredimages(

D

^E⁼⁰

eV)weretakenunderreduceddoseconditions.Allimageswere recorded digitally by a bottom mounted CCD camera system (Ultrascan 1000, Gatan GmbH) and processed with a digital imagingprocessingsystem(GatanDigitalMicrograph3.9forGMS 1.4,GatanGmbH).

2.3.Transfectionagent

The transfection agent was a poly(1,2-butadiene)-block- PDMAEMA (B290D240) block-copolymer as described in [19].

B290D240consistsofahydrophobicpolybutadiene blockwithan average length of 290 monomeric units and a polycationic PDMAEMAblockwithanaveragelengthof240monomericunits.

The number average molecular weight, Mn, of the moleculeis 54kDa,thepolydispersity (Mw/Mn)is <1.07.Stable star-shaped micelles of B290D240 were formed by dissolving the block copolymer in THF and dialyzing the solution against DPBS as described elsewhere [11].B290D240micelles werepreparedasa 10.7mg/mLstocksolutioninsterileDPBS.

2.4.Aggregationnumbercalculation

Aggregation numbers (Nagg)of the micelles werecalculated accordingto:

Nagg¼ mcore

m^chain_PB ¼4

p

^N^Ar^PBR³_core 3M^chain_PB

withmcore=massofthecore,mPBchain=massofonepolybuta- dienechain,NA=Avogadro’sconstant,

r

PB=densityofpolybutadi- ene(=1g/cm³),Rcore=radiusofthecore(asdeterminedbyTEM data),andMPBchain=molecularweightofonepolybutadienechain (=15,700g/mol).

2.5.Celllinesandmaintenance

CHO-K1(adherent,CCL-61,ATCC),Jurkat (humanleukemiaT cells, suspension, TIB-152,ATCC), and HEK-293 (adherent, CRL- 1573,ATCC) cellsweremaintainedinR10,L929cells (adherent, murineﬁbroblast,CCL-1, ATCC)in MEM10,assuggested bythe supplier. Cells werecultivated in thestandard mammalian cell cultureincubator.

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2.6.Plasmids

PlasmidswerepEGFP-N1(4.7kb)encoding fortheenhanced greenfluorescentprotein(EGFP) drivenbythecytomegalovirus (CMV)immediateearlypromoter,pmCherry-N1(4.7kb)encoding for themutant fluorescent proteinderived fromthetetrameric Discosomasp.redfluorescentprotein(mCherry,RFP)drivenbythe immediateearlyCMVpromoter,pZsYellow1-N1(4.7kb)encoding fortheyellowfluorescentprotein(ZsYellow1,YFP)drivenbythe immediateearly CMVpromoter, allfromClontech Laboratories, Inc. (MountainView, CA) and pIVEX23UK (4.4kb, Roche, Man- nheim, Germany) encoding for a mouse urokinase under the controlofthebacteriophageT7promoter.Thelatterwasusedas controlplasmidasduetothechosenpromoterthegeneproductis notexpressedinmammaliancells.Inaddition,plasmidspSV40- EGFP-N1 (4.1kb)encoding for EGFP drivenby thesimianvirus 40earlypromoter(SV40)andpSV40/enhEGFP-N1(4.3kb)encoding for EGFP driven by the SV40 promoter/enhancer were constructed by excising the coding sequence of EGFP out of pEGFP-N1andsubcloning itintothepGL3-promoterand pGL3- controlplasmids(bothfromPromega,Mannheim,Germany)after deletionoftheluciferasecDNAfromtheseplasmids.pEF1

a

^-EGFP-

N1 (7.0kb) encoding for EGFP driven by the elongation factor 1 alpha promoter (EF-1

a

⁾ ^was constructed by subcloning the coding sequenceof EGFPinto plasmidpEAK8(Edge BioSystem, Gaithersburg,MD).

PlasmidswereamplifiedinEscherichiacoli(LBmedium)using standardlaboratory techniques.TheEndoFreePlasmid Kit(Giga Prep/MaxiPrep) fromQIAGEN(Hilden, Germany)was used for purification(qualitycontrol:>80%supercoiledtopology(agarose gel;1%TAE)and A260/A2801.8).Purifiedplasmidsweresolubi- lizedinsterilePCR-water(Sigma-Aldrich).

2.7.N/P-ratiocalculation

N/P-ratioswerecalculatedaccordingto:

Number of equivalent¼

m

^L ^polycation^stock ^solution½N

m

^g ^pDNA3 with[N]=concentrationofnitrogenresiduesinmM.

2.8.Transfection

Fortransfectionofadherentcells(CHO-K1,L929,HEK-293),the cellswereharvested bytrypsinization uponreaching 80%conﬂuency andseededatadensityof210⁵cellsin2mLgrowthmediumper well in six-well plates 24h prior to transfection. CHO-K1 and L929cellswererinsedwithDPBSonehourpriortotransfectionand supplementedwith1mLOpti-MEM.Afterwardstheplatewasput backintotheincubator.Inthemeantime,polyplexesofpDNAand B290D240werepreparedinaﬁnalvolumeof200

m

^L^byﬁrstdiluting thedesired amount ofpDNAstocksolution withHBGbuffertoaﬁnal concentrationofapproximately10

m

^g/mLⁱⁿ^a^1.5^mL^reaction^tube

followedbytheadditionofthenecessaryamountofB290D240stock solution in a singledrop (max. volume: 20

m

^L) ^to^achieve ^the

intendedN/Pratio(7.5forCHO-K1,10forL929cells).Fortheco- transfectionexperiments,theindicatedamountsofbothpDNAs weremixedbeforetheadditionoftheB290D240.Afterwards,the mixturewasvortexedfor10sandincubatedfor20minatroom temperature.1mLOpti-MEMwasadded,followedbyvortexingand 10minincubationatroomtemperature.Thepolyplexmixturewas thenaddeddrop-wisetothecellsanddistributedbygentlyrocking theplate.Cultureswerethenputbackintotheincubator.4hlater themedium was aspirated and replaced by 2mLof freshpre- equilibratedgrowthmedium.

HEK-293 cells (adherent) were transfected using a slightly modiﬁedprotocol(cellsadhereonlyweakly,washingwouldhave ledtoconsiderablelosses).Brieﬂy,HEK-293cellswereharvested andreseededasdescribedaboveforCHO-K1andL929cells,but thenmaintainedunderregulargrowthconditionsrightuptothe timeoftransfection.Thesupernatantwascautiouslyaspiratedand immediatelyreplacedby1.2mLofthepolyplexmixture(N/P-ratio 7.5,drop-wiseaddition).Subsequently,HEK-293cellsweretreated asdescribedabovefortheCHO-K1andL929cells.

For the transfection of Jurkat cells (suspension), cells were harvested bycentrifugation (200g, 5min) from cultures in the exponential growth phase (viability >90%) two hours prior to transfection.CellswerewashedtwicewithDPBS,andseededat 210⁵cellsperwellin1mLOpti-MEM(6-wellplate).Theplate wasputbackintotheincubatorforonehour.Polyplexpreparation andadditiontothecellswereasgivenfortheadherentcells.N/P- ratioswere7.5intheseexperiments.Afterpolyplexaddition,the plates wereplaced intotheincubator for 4hand subsequently centrifuged(200g,5min).Thesupernatantwascarefullyaspirated andreplacedby2mLoffreshpre-equilibratedgrowthmedium.

Transfectionexperimentsusedinthecomparisonswerealways doneinparallelusingonepolyplexpreparation(N/P-ratio7.5);an exception was made for the L929 cells, where a polyplex preparation with an N/P-ratio of 10was prepared instead and used in parallel. N/P-ratios were chosen according to standard protocols previouslyestablished in house for the different cell lines.Ingeneral, transfectionefficienciesincreasewiththeN/P- ratio,whilethecultureviabilitydecreases.Inordertochoosethe N/P-ratio to be used for transfection, the ratio is step-wise increased from the value sufficient to charge-compensate the pDNA (N/P=3 in case of B290D240,as verifiedby zetapotential measurements of the formed polyplexes, Zetasizer Nano ZS, Malvern,Herrenberg,Germany),untileithernofurtherimprove- mentintransfectionefficiencyisobservedorthecultureviability drops below a value of 70%. The fact that polyplexes used for transfection had a positive net-charge was verified by zeta potentialmeasurements.

2.9.Flowcytometry

Forﬂowcytometry(CytomicsFC500,BeckmanCoulter,Krefeld, Germany),adherentcellswereharvestedbytrypsinization(5min), resuspended in the original culture supernatant to include detached/dead cells, recovered by centrifugation (200g, 5min) andresuspendedin500

m

^L^DPBS^containing¹

m

^g/mL^propidium

iodide(PI)tocounterstainthedeadcells(exceptcellstransfected withpmCherry-N1,wheretheredﬂuorescencecausedbyPIwould have beenindistinguishablefromthat of mCherry).Jurkatcells weredirectlyrecoveredbycentrifugationandresuspendedinthe 500

m

^L^DPBS^containing^the^PI.^Forward^scatter^(FSC),^side^scatter

(SSC), green fluorescence (em 510nm), red fluorescence (em 620nm)andyellowfluorescence(em550nm)wererecorded.For this,theflowcytometerfilterblockconfigurationwasmodifiedto allow thesimultaneous optical separation of green and yellow fluorescence (EGFP/YFP), which cannot be separated with the standardfiltersconfigurationdueanextensivespectraloverlap.To assurecomparabilitybetweentheobtaineddata,carewastakento usethesameinstrumentsettingsinallexperimentsinvolvinga particularreporterprotein.Cellswereinitiallyevaluatedbyscatter properties(FSC/SSC)inordertoselectaregionrepresentingsingle, non-apoptotic cells, while disregarding dead cells, debris and cellularaggregates.Cells‘transfected’atN/P=0,i.e.intheabsence of the transfection agent, were used to set the measurement parameters. Histogram plots of the respective fluorescence intensities (log scale)were usedto estimatethe percentage of transfected cells and the expression strength distribution

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accordingto:lowproducers (L):fluorescenceintensitybetween 1 and 10 a.u.; middle producers (M): fluorescence intensity between 10 and 100 a.u.; high producers (H): fluorescence intensity>100a.u.)inthenon-apoptoticcellpopulation;witha.

u.:arbitraryunits.

2.10.Statisticalanalysis

GroupdataarereportedasmeanSD. One-wayANOVAwas usedtodeterminewhetherdatagroupsdifferedsignificantlyfrom eachother.Statisticalsignificancewasdefinedasp<0.05.

3.Resultsanddiscussion 3.1.Thetransfectionagent

Toexcludeanyinﬂuenceofthetransfectionagent,experiments wereexclusivelyconductedwithB290D240,a polycationicagent, which haspreviouslybeendescribed forits ability totransfect siRNA into CHO cells [11]. In aqueous solution B290D240 forms stablestar-shapedmicelleswiththepolybutadieneblockforming ahydrophobic inner core,as conﬁrmedbycryogenic TEM.This micellar structure can therefore be considered a somewhat unusual yet functional representation of the general design principle of a star-shaped polycationic agent, which otherwise [11,12] has been embodied in the form of polycationic arms covalentlygrown froma solid centralcore. B290D240 is by now routinelyusedfortransfectioninourgroup,asitgivessuperior resultsforalargenumberofcells,someofwhichareincludedin thispublication.

Evaluationofapproximately100micellesbyimageanalysisof TEM data allowed calculating an average core diameter of Dcore=18.22.5nm, i.e. a core radius Rcore of 9.1nm, for the micelles.Under theassumption thatthecoreofthemicelles is sphericalandconsistsonlyofpolybutadiene,anaverageaggrega- tionnumberNaggof120wascalculatedforthemicelles.Compared, e.g. to the gold standard among the polycationic transfection agents,namelyPEI,B₂₉₀D₂₄₀hasaverylowpolydispersity(<1.1), whileaccordingtotheTEMdata,thecorrespondingmicelleshavea narrowsizedistribution.Thisshouldreducethecontributionofthe transfectionagent’sheterogeneitytothevariabilityoftheexperi- ments.

3.2.Inﬂuenceofthepromoter

Theinfluenceofthepromoteronthetransfectionoutcomefora givencelllineunderotherwiseidenticalexperimentalconditions wastestedusingtheenhancedgreenfluorescentprotein(EGFP)as reportergeneproduct.Thetransfectionefficiency(percentageof cells expressing the transgene within the population) and the distribution of the expression strength (high, middle, and low producers)amongthesuccessfullytransfectedcellswasanalyzed 48hposttransfection.ResultsaregiveninFig.1togetherwiththe cultureviabilities.

TransfectionefﬁcienciesinFig.1varyconsiderablyinspiteof thefactthatthesametransfectionagentandconditionshadbeen used. For a given transfection agent some dependency of the transfectionefﬁciencyontheoncelltypehastobeexpected.The promoter,ontheotherhand,shouldhaveaneffectonthestrength ofthetransgeneexpression.Thisis,e.g.,seenincaseoftheCHO- K1 cells for thethree viralpromoters. Expression was weakest under the control of the SV40 promoter (Fig.1, bar B). Better expressionwas obtainedin the presenceof the SV40/enhancer (Fig.1,barC).TheCMVpromoterwasbyfarthemosteffectivein theCHO-K1cells,leadingtoalmost80%ofhighproducers(Fig.1, bar D). Interestingly, the human cellular promoter EF-1

a

performed at least as well as the SV40/enhancer in the CHO- K1cells(Fig.1,barA).

Apromotereffectonthetransfectionefﬁciency,i.e.DNAuptake, wasnotexpected,sincefora givencelltype/transfectionagent, DNAuptakeshouldbeindependentofthesequencedetailsofthe transfectedDNA.Ifanything,theplasmidsizehasinthepastbeen showntoaffectDNAuptake.However,thiswasnotthecasehere, wherethepercentageoftransfectedcellswassimilarforpEF1

a

^-

EGFP-N1(7.0kb)andpSV40-EGFP-N1(4.1kb)orpSV40/enhEGFP- N1(4.3kb).Thesigniﬁcantlyhigherpercentageoftransfectedcells observedinallexperimentsinvolvingtheCMVpromoterthushas to benoted. It is possible that this is an artifact and that the apparenthigherpercentageoftransgeneexpressingcellsfoundfor theCMVpromoterisinsteadsimplyrelatedtoastrongertransgene expression.OnlyafewlowproducerswereobtainedwiththeCMV promoter.Fortheotherpromotersthelowproducerfractionwas much larger. It is possible that in such cases some very low producersarelostduringgating,therebyreducingthepercentage of transfected cells. However, a direct effect of the viral CMV promoteronDNAuptakeinadditiontogeneexpressioncannotbe excludedatthispoint.

Trendsobservedinthesecondrodentcellline(L929)forthe promotereffectontransfectionefﬁciencyandexpressionstrength weresimilar,withtheexceptionofthehumancellularpromoter EF-1

a

^,^for^which^the^lowesttransfectionefﬁciencywasobservedin case of the L929 cells. This we interpret as a size effect. As mentioned above, plasmidsize differencesare knownto cause differencesintransfectionefﬁciency.

Inthetwohumancelllines,theCMVandthehumancellular promoter EF-1

a

^show â ^similar distribution of the expression strengthoverhigh,middleandlowproducers,butthetransfection efficiencyisagainsignificantlylowerincaseofthelargerpEF1

a

^-

EGFP-N1plasmid.Ifonecomparestheperformanceofthethree viralpromotersinthehumancelllines,expressiondrivenbythe SV40promoterisagainweakerthanfortheCMVpromoter,while

viability [%]: 72 74 73 74 84 86 85 86 87 88 85 89 97 99 98 93

transfected cells [%]

0 20 40 60 80 100

promoter type: A B C D A B C D A B C D A B C D cell line: Jurkat HEK-293 L929 CHO-K1

***

**

***

Fig. 1.Promoter effect on transfection efficiency and expression strength distribution.EGFPexpressionunderthecontrolofthecellular(A=pEF1a-EGFP- N1)andthethreeviral(B=pSV40-EGFP-N1,C=pSV40/enhEGFP-N1,D=pEGFP-N1, i.e.CMV)promoterswasquantified48hposttransfection.Theheightofthebar correspondstotheoveralltransfectionefficiency,i.e.thepercentageoftransgene expressingcellswithinthepopulation.Thetransfectedcellpopulationwasfurther dividedinto:lowproducers(white,fluorescencesignalbetween1and10a.u.), middleproducers(grey,fluorescencesignalbetween10and100a.u.),andhigh producers(darkgrey,fluorescencesignal>100a.u.).Groupdataarereportedas means.d.fromthreeormoreindependentexperiments.Statisticalsignificanceis indicatedby*(p<0.05).

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contrarilytotherodentcells,expressionstrengthisnotimproved in the human cells by the presence of the SV40/enhancer. In additiontothe–expected–inﬂuenceontheexpressionstrength, however,inthehumancellsaswellweseeapronouncedpromoter effect in the total transfection efﬁciency between the CMV promoterandthethreeothers,arguingoncemoreforapromoter effectonnon-viralDNAuptake.Incidentallyweneverobserved anysilencingoftheCMVpromoterasobservedbyQuinetal.[20]

inourexperiments.

3.3.Inﬂuenceofthetransgene

Inordertoinvestigateputativeeffectsofthereportertransgene, twoadditionalfluorescentproteins,namelyYFP(yellowfluores- centprotein) andRFP (redfluorescentprotein) wereexpressed underthecontroloftheimmediateearly CMVpromoterin the investigated cell lines. Experiments were again performed in parallelforthefourcelllineskeepingexperimentalconditionsas similaraspossible.ResultsaresummarizedinFig.2.Sinceitisnot possibletodistinguishbetweenthefluorescenceofPIandRFP,cell viabilitiescouldonlybedeterminedforcellstransfectedwithEGFP andYFP.However,alldeterminableviabilityvalueswerein the samerangeasthoseobservedbeforeandweassumethistobethe caseforRFP-transfectedcellsaswell.

Transfection efficiencies in these experiments showed no statisticallyrelevantvariationinHEK-293andCHO-K1cells,while thecriterionforstatisticalsignificance,definedasp<0.05, was justslightlysurpassedincaseoftheL929cells.Thisresultisnot surprising since the three vectors belong to the same plasmid familyandthereforehaveasimilarbackbone/size.

However, in spite of the fact that the same strong (CMV) promoter was used to drive expression in all three vectors, differencesin the expression strength wereobserved. Whereas EGFPandYFPshowedasimilardistributionoverlow,middleand highproducers,RFPexpressionwaslessstronginallinvestigated celllines(almostnohighproducers,mainlylowproducers).Codon

usageincaseofEGFPhasbeenoptimizedformammalianandin case of YFP even forhuman cells. Thisis not thecase for RFP.

However,itisunlikelythatthelowerexpressionobservedforRFP isduetosuboptimalcodonusage,aswasverifiedbyanalysisofthe cDNAsequence[21].Problemswithincompletematuration,which have been reported for the tetrameric parent protein DsRed [22,23], should not occur for the monomeric mutant mCherry (RFP),whichhasbeenreportedtohaveahalftimeformaturationof 10min(DsRed:10h).However,sinceweconsistentlyobserve‘low RFPexpression’inallinvestigatedcelllines,itispossiblethatthisis simpleduetodifficultiesindetectingtheredfluorescence,i.e.a lower ‘brightness’ of the protein. According to Shaner et al., mCherryisalsomoresensitivetophotobleachingthan,e.g.,EGFP [24].

Whileaconsistentlylowerstrengthoftransgeneexpressionin thecase ofmCherrycanthereforebeattributedtothereporter protein itself, this is not the case for the transfection results obtained for the Jurkat cells. While the comparatively low transfectionefficienciesfoundforEGFP(50%)andRFP(34%)were withintherangeseenaboveforEGFPexpressionundertheCMV promoter, transfection of the Jurkat cells with YFP was almost impossible(transfectionefficiencies<20%).Itshouldbenotedthat in spiteof thelowoveralltransfectionefficiency,theexpression strength distribution of the few YFP-transfected Jurkat cells showed a significant fractionof highproducers. In fact, in the Jurkat cells therelative size (percentage) of the highproducer fractionwashigherfor YFPthanfor theothertwoinvestigated fluorescentproteins.Theoptimizedcodonusagemightbeinthat caseresponsibleforamoreefficienttranslationofYFPinhuman cells.ThuswithpZsYellow1-N1wehaveanexampleforaplasmid which in spite ofa similar backboneis much more difficultto transfect intoJurkat cells than pEGFP-N1 or pmCherry-N1, but whichinthefewsuccessfullytransfectedcellsisexpressedwith superiorstrength.

Moreover,inspiteoftheirsizeB290D240micelleswerefoundto beverybiocompatible.Inparticular,thedirectcorrelationbetween transfectionefﬁciencyandcytotoxicityreportedformostnon-viral transfectionagents wasnot seenin thiscase. EvenJurkatcells, which are known to be sensitive to polycationic transfection agents, showed viabilities >70% post transfection, whereas the valuesfortheotherinvestigatedcelllineswereroutinelyabove 80%(>90%incaseoftheCHOcells).

3.4.Pair-wiseco-transfectionofthereportergenes

Experiments involving more than one reporter proteinhave significance beyond the investigation of promoter/transgene effects.However,asfaraswecouldascertain,theco-expression oftwofluorescentproteinsinmammaliancellshassofarnotbeen studied in a systematic manner. Zhu et al. [25] reported the simultaneousdetectionandquantificationofEGFP,YFP,andCFP (cyan fluorescent protein) expressed from di- and tricistronic constructsaftertransfectionwithLipofectamineinHEK293cells.

However, the authors only gave the MFI (mean ﬂuorescence intensity) for the cultures and did not discriminate between transfected and non-transfected cellsor low,medium andhigh producers.

Inordertoinvestigateeffectsinco-transfection,theplasmids encodingforthethreeﬂuorescentproteinsunderthecontrolofthe CMV promoterwere transfected pair-wise intothe cells. Equal amounts(byweight)ofplasmidwereused,whilekeepingthetotal amountofplasmidDNAthesameasinthepreviousexperiments.

TheresultsaresummarizedinFig.3.Inthisﬁgurethepercentage ofbichromatographiccells withinthepopulationaswellasthe percentagesofcellsexpressingonlyoneoftherespectivereporter proteins (“monochromatic” cells) are shown. Within each

0 20 40 60 80 100

cell line: Jurkat HEK-293 L929 CHO-K1

#

*

G Y R G Y R G Y R G Y R

Fig. 2.Transgene effect on transfection efficiency and expression strength distribution.ExpressionofEGFP(G,diagonaltoprightlines),YFP(Y,diagonal topleftlines)andRFP(R,horizontallines)allunderthecontroloftheintermediate earlyCMVpromoterwasquantified48hposttransfection.Theheightofthebar correspondstotheoveralltransfectionefficiency,i.e.thepercentageoftransgene expressingcellswithinthepopulation.Thetransfectedcellspopulationwasfurther dividedinto:lowproducers(white,fluorescencesignalbetween1and10a.u.), middleproducers(grey,fluorescencesignalbetween10and100a.u.),andhigh producers(darkgrey,fluorescencesignal>100a.u.).Groupdataarereportedas means.d. from three or more independent experiments and the statistical significanceisindicatedby*(p<0.05).Statisticalsignificancewithinacelllineis indicatedby#(p<0.05).

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subpopulation, the expression strength is again indicated by shading.Please notethat in this ﬁgurethe overalltransfection efﬁciencycorrespondstothesumofall transfectedsubpopula- tions, i.e. the percentage of bichromatographic cell plus the percentage of monochromatic cells of subtype 1 and the

monochromaticcellsofsubtype2.Subpopulationswithastrength 1%arenotshowninthispresentation.

For all investigated cells, except the Jurkat cells, total transfection efﬁciencieswere highest for cells transfected with EGFP/YFP, where they were roughly in the same order of CHO-K1

GFP YFP GFP YFP

0 20 40 60 80 100

GFP RFP GFP RFP YFP RFP YFP RFP monochromatic

cells bichromatic

cells

monochromatic cells bichromatic

cells

HEK-293

GFP YFP GFP YFP

0 20 40 60 80 100

cells bichromatic

cells

Jurkat

GFP YFP GFP YFP

0 20 40 60 80 100

cells bichromatic

cells

L929

GFP YFP GFP YFP

0 20 40 60 80 100

cells bichromatic

cells

Fig.3.Co-transfectionexperiments,involvingpair-wiseapplicationoftwoencodingplasmidDNAs.Equalamounts(byweight)ofpDNAencodingfortherespectivereporter proteinsunderthecontroloftheintermediateearlyCMVpromoterwereused,withthetotalamountofpDNAequalingthatusedintheexperimentsinvolvingonlyone reporterproteinunderotherwiseidenticalexperimentalconditions.ExpressionofEGFP(diagonaltoprightlines),YFP(diagonaltopleftlines)andRFP(horizontallines)under thecontroloftheintermediateearlyCMVpromoterwasquantified48hposttransfection.Indicatedarebichromaticcellsexpressingbothproteinsandmonochromaticcells expressingonlyoneoftheproteins.Transfectedcellspopulationwasfurtherdividedinto:lowproducers(white,fluorescencesignalbetween1and10a.u.),middleproducers (grey,fluorescencesignalbetween10and100a.u.),andhighproducers(darkgrey,fluorescencesignal>100a.u.).

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magnitudeasintheexperimentsinvolvingonlyEGFP,seeFig.1for comparison. In case of the EGFP/RFP transfection, overall transfection efﬁciencies were slightly lower, while the lowest values were obtained in the experiments involving YFP/RFP.

Moreover, instead of obtaining only – or at least mainly – bichromatic cells, the co-expression experiments unexpectedly resultedinsigniﬁcantnumbersofmonochromaticcells.Ingeneral, thepropensityforco-expressiondependedonboththecelltype and the reporter protein(s). For instance, the CHO-K1 cells consistently showed the highest co-expression rates (80% of thetransfectedcells),whereasthisfractioncouldbeaslowas30%

incaseoftheJurkatorL929cells.Intheco-expressionexperiments involvingEGFP,themajorityofthemonochromaticcellsexpressed EGFP and not the respective other protein. In the YFP/RFP experiments, all investigated cells, except for the Jurkat cells, expressed either both proteins or only YFP. The fraction of monochromatic RFP-expressing cells was less than 2% of the transfectedcells.

ResultsfromtheJurkatcells,ontheotherhand,pointagain towards a general difﬁculty with YFP. In the EGFP/YFP-co- transfectionsanunusualhighfraction (>70%)ofthetransfected

cellsweremonochromaticandexpressedEGFP,whileintheEGFP/

RFPco-expressionexperimentsthepercentageofmonochromatic (green)cellswasonly50%ofthetransfectedcells.Moreover,the Jurkatcellsweretheonlyones,whereasigniﬁcantnumberofthe transfectedcells(>40%)expressedonlytheredﬂuorescentprotein afterco-transfectionwithYFP/RFP.

Whencellsexpressingoneorbothfluorescenceproteinswere furtherclassifiedintohigh, middle,andlowproducers,Fig.3,it becameclearthathighproducersweremainlyfoundamongthe bichromaticcells,whereasthemonochromaticcellsfromthesame populationtendedtobemiddleandlowproducers.Thuscellsthat stronglyexpressedthefluorescentproteinsoftenwereabletoco- expressboth proteins,whiletheinabilitytoco-expressthetwo fluorescentproteinscoincideswithalowoverallexpressionlevel.

3.5.EffectofpDNAdilution

If one considers transfection efﬁciencies per individual transgene, for EGFP similar levels were reached in the co- transfection experiments in spite of the fact that only half as much pDNA had been used per individual reporter protein

pDNA dilution: A B C D A B C D A B C D

0 20 40 60 80 100

pEGFP-N1 pZsYellow1-N1 pmCherry-N1 HEK-293

I

pEGFP-N1 pZsYellow1-N1 pmCherry-N1 Jurkat

II

0 20 40 60 80 100

**

0 20 40 60 80 100

pEGFP-N1 pZsYellow1-N1 pmCherry-N1 L929

*

**

III

0 20 40 60 80 100

pEGFP-N1 pZsYellow1-N1 pmCherry-N1 CHO-K1

*

IV

Fig.4.EffectofpDNAdilutionontransfectionefficiencyandexpressionstrengthdistribution.(I)HEK-293,(II)Jurkat,(III)L929,(IV)CHO-K1.ExpressionofEGFP(diagonaltop rightlines),YFP(diagonaltopleftlines)andRFP(horizontallines)underthecontroloftheintermediateearlyCMVpromoterwasquantified48hposttransfectionwith polyplexescontainingdecreasingamountsofencodingpDNA.FordilutionofthespecificpDNAatconstanttotalpDNAinthetransfectioncocktail,polyplexeswereprepared replacing0,25,50,and75wt%(A–D)oftheencodingDNAbyanon-expressedcontrolplasmid(pIVEX23UK).Transfectedcellswerefurtherdividedinto:lowproducers (white,fluorescencesignalbetween1and10a.u.),middleproducers(grey,fluorescencesignalbetween10and100a.u.),andhighproducers(darkgrey,fluorescencesignal

>100a.u.).Groupdataarereportedasmeans.d.fromthreeormoreindependentexperiments.Statisticalsigniﬁcanceisindicatedby*(p<0.05).

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comparedtotheexperimentsinvolvingonlyonetypeofpDNA.For example,theCHO-K1cellpopulationinFig.3co-transfectedwith pEGFP-N1 and pZsYellow1-N1 contained a total of 83% EGFP positivecells (66.7%bichromaticcells expressingboth proteins, 15.1% monochromatic ones expressing only EGFP). It is thus unlikelythatthereductioninexpressionobservedfortheother twoﬂuorescentproteinsintheco-transfectionexperimentswas duetothefactthatlessencodingpDNAwastransfectedinthese experiments.Suchareductioninproteinexpressionasafunction oftheamountoftransfectedplasmidhas,e.g.beenobservedbyLiu etal.[26].Instead,sinceinourcaseallproteinswereunderthe controlofthesame–strong–CMVpromoter,a“saturationeffect”, e.g.duetocompetitionforsometranscriptionfactors,couldnotbe excluded.

Subsequently, we therefore analyzed the effect of specific plasmidcontentinatransfectioncocktail.Asimplereductionof thepDNA amountwas notsuitable for this purpose, since this wouldhaveaffectedtheN/P-ratio,whichisknowtoaffectboth transfectionefficiencyand culture viability[27]. Addingvaried amountsofpolyplexestothecellswasalsonooption.Instead,a pDNA (pIVEX23UK) encoding for a mouse urokinase underthe controloftheT7promoter,wasusedtodilutethepDNAsencoding for the various fluorescent proteins. T7 is a bacteriophagic promoter and requires the presence of the bacteriophage T7 RNA polymerase for activity. Unless mammalian cells are geneticallymodifiedtoexpressthispolymerase,whichwasnotthe caseinourexperiments,thispromoterisnotactive[28,29].The presenceofpIVEX23UK shouldthereforenot interferewiththe expressionof EGFP,YFP or RFP bythecells. Theresultsof the experiments are summarized in Fig. 4. Bars A–D represent experimentsinwhich0,25,50,or75 wt%of thespecificpDNA hadbeenreplacedbypIVEX23UK.In otherwords, barAcanbe directlycomparedtotheexperimentssummarizedinFig.1,while thespecificplasmidamountsintheexperimentscorrespondingto bar C were identical to the ones used in the experiments summarizedinFig.3.

Somedifferencesareagainobservedasafunctionofthecelland transgene type. CHO-K1 cells, for example, showed constant transfection efﬁciencies independent of the dilution, with the possibleexceptionofperhapstheverylowestplasmidcontent(25 wt% of the original, bar D), where a statistical difference in transfectionefﬁciencycanbeobservedatleastforpZsYellow1-N1.

The expression strength distribution, on the other hand, was increasinglyshiftingawayfromthehighproducersintheCHOcell experimentsastheplasmidwasdiluted.Thus,thenumberofcells thattakeuptheplasmidseemstobesimilarregardlessofdilution, but highproducersare more likely tooccur when thespeciﬁc plasmidisnotdiluted.Thiswouldargueagainstthehypothesisofa transcription factor shortage at higher plasmid concentrations, sincemoreplasmidobviouslystillresultsinmoreprotein.Hence suchashortageisalsounlikelytobedirectlyresponsibleforthe decreased protein expression in some of the co-expression experimentsdiscussedabove.

In the HEK-293 cells, on the other hand, neither the transfection efﬁciency nor the expression strength is affected bytheplasmiddilution,exceptperhapsincaseofRFP,wherethe fractionofhighandmiddleproducersdiminishesincaseofthe highest plasmid dilution (bar D). A similar behavior, i.e.

comparabletransfectionefficiencyandexpressionleveldistribu- tionexceptforthehighestdilution,isobservedfortheL929cells, butinthiscaseforallthreefluorescentproteins,whileJurkatcells consistentlyshowastatisticallysignificantdecreaseintransfec- tion efficiency, but not in the expression strengthdistribution withdilution.Amechanisticinterpretationoftheseobservations wouldrequireadetailedstudyoftheintracellularevents,which isfarbeyondthescopeofthiswork.However,aclearconclusion

canbe drawnregardingthenecessity tocarefully evaluatethe applicability of ﬂuorescence reporter proteins (pairs) for any givencellularsystem.

4.Conclusions

Theexpressionofarecombinantproteindependsforagiven celllineandtransfectionprotocolonboththepromoterandthe transgenesequence.Nogeneraltendenciescouldbeobservedfor this behavior in our experiments, not even among the two investigatedhumanandrodentcelllines,respectively.‘Standard’ conditionssuchasCMVpromoter,EGFPasreportergeneproduct, andthechosenstandardtransfectionprotocolworkedwellforthe CHO-K1cellsforwhichtheyhad beenoriginallydeveloped,but werenotalwaysequallysuccessfulfortheotherinvestigatedcells.

Moreover, based on past experience in our group with the developmentoftransfectionprotocols,theeffectachievablewith optimizingagivenprotocolismuchsmallerthanthedifferences observedhereasafunctionofthepromoter/transgenetype.We thereforeproposethatanydevelopmentofatransfectionprotocol for mammalian cells should start with a chemometric multiparameter investigation involving cell type, promoter type, transgene,andlastbutnotleastalsothetransfectionagent.This aspectwasnotincludedinourinvestigation,butitislikelythatnot alltransfectionagentsshowsimilarperformanceforallcelltypes.

Conﬂictofinterest

Theauthorsdeclarenoconﬂictofinterest.

Acknowledgements

This work was funded by the Upper Franconian Trust (Oberfrankenstiftung, Bayreuth, Germany) grant P-Nr.: 03847.

UllrichStahlschmidtclonedtheadditionalplasmids,whileAndrea Schott and Philipp Neßbach supported this study by purifying someoftheplasmids.

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