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Efficient Lipid-Mediated Transfection of DNA into Primary Rat Hepatocytes Author(s): Sheri L. Holmen, Matt W. Vanbrocklin, Robert R. Eversole, Susan R. Stapleton and Leonard C. Ginsberg Source: In Vitro Cellular & Developmental Biology. Animal, Vol. 31, No. 5 (May, 1995), pp. 347351 Published by: Society for In Vitro Biology Stable URL: http://www.jstor.org/stable/4294424 . Accessed: 22/04/2013 12:35 Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at . http://www.jstor.org/page/info/about/policies/terms.jsp

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In Vitro Cell. Dev. Biol. 30:347-351, May 1995 ? 1995 Society for In Vitro Biology 1071-2690/95 $05.00 + 0.00

EFFICIENT LIPID-MEDIATED TRANSFECTION OF DNA INTO PRIMARY RAT HEPATOCYTES ROBERTR. EVERSOLE,SUSAN R. STAPLETON,and LEONARDC. GINSBERG' SHERIL. HOLMEN,MATTW. VANBROCKLIN, Departmentof Biological Sciences,and Departmentof Chemistry,WesternMichigan University,Kalamazoo,Michigan49008 (Received 23 September1994; accepted 6 December1994)

Cationic lipids are an effective means for transfecting nucleic acids into a variety of cell types. Very few of these lipids, however, have been reported to be effective with primary cells. We report on the efficacy of several commercially available cationic lipid reagents to transfect plasmid DNA into primary rat hepatocytes in culture. The reagents tested in this study include TransfectAce, LipofectAmine, Lipofectin, N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammoniumchloride (DOTMA), (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium methylsulfate (DOTAP), and cetyltrimethyl-ammonium bromide/dioleoylphosphatidylethanol-amine (CTAB/DOPE). Electron micrographic (EM) studies indicate that similar size Lipofectin and DOTAP vesicles contain DNA-like material internally and that these vesicles attach to the cell membrane. DOTAP vesicles are multilamellar, appear as clusters, and have a high DNA-to-lipid ratio. Lipofectin vesicles appear to attach to the cell surface as individual vesicles. The EM observations are consistent with current theories on the mechanism of transfection by cationic lipids. While Lipofectin has proven to be effective in transfection studies of primary cells in culture, we have found DOTAP to be a viable alternative. DOTAP yields transfection rates in hepatocytes comparable to DOTMA and Lipofectin, however, at lower concentrations of reagent and at considerably less cost. Optimal of DNA with DOTAP were achieved by utilizing multilamellar (vortexed) vesicles conditions for transfecting 5 lag plasmid at a concentration of 15 DOTAP per 2 ml media in 60-mm plates for 2 h transfection time. In this study, DOTAP has easy to prepare, and very effective in transfecting DNA into primary rat hepatocytes. proven to be economical, -tg Key words: DNA transfection; cationic lipids; primary hepatocytes. INTRODUCTION The ability to introduce DNA into mammalian cells in culture provides a powerful means for studying the function and control of mammalian gene transcription. The use of primary cells in culture for these studies offers the advantage of studying cells in a metabolic and differentiated state close to that found in vivo. A wide variety of methods have been developed to transport DNA into cells. While a number of these techniques have been very effective in the transfection of nucleic acids into cells from established lines, cationic lipid reagents remain the most widely used method for transfection of DNA into primary cells in culture (6,13). Previous studies have shown that the lipid reagents DOTMA and Lipofectin are effective in transfecting primary hepatocytes in culture (13). Recently, a number of other lipid reagents have also become available. These reagents have been extensively tested on a number of established lines to demonstrate their efficiency of DNA transfection (1,2,3,7,8,9,10,14,17). However, little is known about the effectiveness of these reagents in mediating transfection of DNA into primary cells. Because it is not possible to make transfected stable lines with primary cells, the cost of the commercially prepared transfection reagents can significantly increase experimentation cost and influTo whom correspondenceand reprint requests should be addressed at Departmentof Biological Sciences, Western MichiganUniversity,Kalamazoo, Michigan49008.

ence the experimental protocol. Therefore, there is a need to find commercially available reagents that not only have a high efficiency of transfection with primary cells but that are also cost effective. We report here on the efficiency of several commercially available reagents to transfect plasmid DNA into primary hepatocytes. ANDMETHODS MATERIALS Cationiclipids. The lipids used include: DOPE(Fluka, Ronkonoma,NY), CTAB (Sigma Chemical Co., St. Louis, MO), TransfectAceT", LipofectAmine'", Lipofectin@ (GIBCOBRL,Gaithersburg,MD),DOTAP(AvantiPolar Lipids, Alabaster,AL), and DOTMA(a gift from D. MontgomeryBissell, Universityof Californiaat San Francisco, Liver Center Laboratory).Transfectace reagent is a 1:2.5 (wt/wt)liposome formulationof the cationic lipid dimethyl dioctadecylammoniumbromide (DDAB) and the neutral lipid DOPE. Lipofectaminereagent is a 3:1 (wt/wt)liposome formulationof the polycationic lipid 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-ditrifluoroacetate methyl-1-propanaminium (DOSPA)and DOPE.Lipofectinreagent consists of a 1:1 (wt/wt)mixtureof DOTMAand DOPE. Hepatocyteisolationand culture.Hepatocyteswere isolatedfrom8-12-wkold, male Fisher rats (HarlanSprague-Dawley,Indianapolis,IN) by in situ perfusion of the liver with collagenase and hyaluronidaseas described in Stapletonet al. (16). Culturesubstrataconsisted of type I collagen from rat tail. The hepatocyteswere plated on 60-mm collagen-coatedculture dishes to a confluencyof approximately90%. Cultureswere incubatedin 4 ml Waymouth'sMB 752/1 medium(GIBCOBRL, Gaithersburg,MD)with 0.5% bovine serum albumin(BSA)at 370 C, 5% CO2:95% air. The media was supplementedwith gentamicin.After6 h, the media was changedto serum-free Waymouth'smedium,and allowedto incubate overnight. Plasmid DNA. The plasmid DNA used for analysis of the efficiency of transfectioncontains 935 bases of the rat glucose-6-phosphatedehydroge-

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HOLMEN ET AL. TABLE1 COMPARISON OF RELATIVETRANSFECTION EFFICIENCIESOF TESTEDREAGENTSa

Transfection Agent

Lipofectin DOTAP LipofectAmine TransfectAce CTAB/DOPE DOTMA

Optimum Transfection Concentration"

Relative Efficiency As Compared to DOTAP

Relative Cost per Plate As Comparedto DOTAP

1.0 1.0 0.2 0.1 0.0c 1.0

140 1.0 90 290 .01 NAd

25 glg 15 pg 25 pLg 70 jlg 25 jlg 25 pg

Hepatocyteswere transfectedwith 5 jlg of plasmid DNA containingthe luciferase reportergene, and the indicatedamountof reagentfor 6 h. Cells were processed 36 h fromthe startof transfection.Relative values are fromat least two differenthepatocyteisolations. b Amountused per 2-ml media in a 60mm plate. d Not commerciallyavailable. Insignificantvalue because it was too low to measureaccurately.

nase (G6PDH) promoterregion (11) inserted between the SmaI and XhoI restrictionsites 5' of the luciferasegene in the p-GL2Basic Vector(Promega, Madison,WI).Fortransfection,plasmidDNAswereisolatedfromthe bacteria by alkaline lysis (15) and purifiedpriorto transfectionusing an ion exchange column (The Nest Group,Southboro,MA).The purityof the DNA was confirmedby agarosegel electrophoresisand A260/A280 absorptionratios. Preparationof vesicles and transfection.Lipofectin, LipofectAmine,and TransfectAcewerepurchasedas vesicle suspensionsin sterile water.DOTAP vesicles were preparedby transferring1.0 mg of a chloroformsolution of DOTAPto a small glass vial and dryingthis under vacuumfor at least 2 h at roomtemperatureto createfilmon the glass vial. One ml of doubledistilled waterwas added to the vial and mixed by vortexingat high speed for 15 s, or by waterbath sonicationfor 5 min. CTABwas combinedwith DOPE(1:4) in chloroform,and dried under vacuumfor at least 2 h as described above. The CTAB/DOPEmixturewas hydratedto a final concentrationof 1.0 mg/ ml with phosphate-bufferedsaline (PBS)and sonicated for 1 min (10). The cells were transfectedwith a complexconsistingof plasmidDNA and the transfectionreagent,6 h or 18 h after plating. Five plgof plasmid DNA per plate was complexed with the transfectionreagent, diluted to a volume of 250 pl with serum-freemedium, and allowed to sit for 15 min at room temperature.The concentrationsof lipid used are specified in Table 1. This complex was then added to the cells in culture and incubated in 2 ml of serum-free Waymouth'smedium for the times specified. The cells were washedwithfreshserum-freemediumand reincubatedin serum-freemedium for 36 h fromthe startof transfectionto allow for expressionof the plasmids. The cells were then scraped,lysed, and analyzedfor the presence of luciferase using a scintillationcounteras recommendedby Promega(4). Luciferase activity is reportedas CPM/plgof cellular protein in the sample. Each data pointrepresentsthe meanof duplicateor triplicateobservationsplus or minus standarderrorof the meanfromat least twodifferenthepatocytepreparations. Statistical analysis was done using MicrosoftEXCEL4.0. Media from the harvested cells was analyzed for lactate dehydrogenase(LDH) activity to assess toxicity of the reagentsto the culture. Electronmicroscopy.The hepatocyteswere fixed in the tissue culturedish as a monolayer2 h after addition of the DNA-lipid complex. The fixation protocolconsisted of primaryfixationwith cold 3% glutaraldehydein 0.1 M sodium cacodylatebuffer(pH 7.2) for 1 h, and postfixationwith 1% osmium in 0.1 M sodium cacodylatebufferovernightat roomtemperature.The cells were dehydratedin a graded series of ethanol and then immersedin a 1:1 mixtureof ethanol:Polybed812 (Polysciences,Warrington,PA) for 1 h. One ml of Polybed 812 was added to the tissue culture dish and allowed to polymerize at 700 C for 72 h. Ultrathin(60-90 nm) sections were cut with a diamondknife using the LKBUltrotomeNovaUltramicrotome,and collected on 200 mesh copper grids. The grids were stained with methanolicuranyl acetate and Reynold'slead citrate, and viewed on a Siemens 101 Transmission ElectronMicroscope. RESULTSANDDISCUSSION Transfectionefficiency.A large numberof cationic lipids have been synthesized for use in DNA transfection(1,2,3,6,7,8,12,13,14). Because the exact mechanism of transfectionby cationic lipid reagents is unknown,the usefulness of these lipids in mediatingDNA transfectioncan only be deter-

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Fi;. 1. Comparisonof reagentsused to transfectprimaryrathepatocytes. Hepatocyteswere transfected18 h after isolationwith variouslipid reagents complexedwith 5 jlg of plasmid DNA. The plasmidcontainsthe luciferase reportergene under the controlof the promoterfor G6PDH.Cells were kept in the reagentfor6 h and assayedforluciferaseactivity36 h aftertransfection was begun. Luciferaseactivity is reportedas counts per minute(CPM)per jLgof cellular protein in the sample. The results are means of duplicate or triplicateplates fromat least threehepatocyteisolations.Errorbarsrepresent the standarderrorof the mean.

minedby testing the reagentsin vitro. Very few of these reagentshave been tested on primarycells. reagent Jarnaginet al. (6) foundthat DOTMAand the DOTMA-containing Lipofectinwereeffectivein transfectingprimaryhepatocytes.Theyalso found that a reagentcomposedof DOTAP/DOPEand TransfectAcegave very low ratesof transfection.These resultsare consistentwithourfindings.We found, however,that DOTAPby itself was an efficienttransfectionreagentas measured by the comparableamountof luciferase activity/pgof cellular protein (Fig. 1). Table 1 shows the optimumconcentrationfor the reagentstested, and the relative efficiencyof the reagentsas comparedto DOTAP.Optimum concentrationswere those that gave the highest level of luciferaseactivity withoutincreasingmedia LDHactivitymorethan 15%. LDHwas used as an indicatorof cellular damageand toxicity caused by the reagentstested. Efficiency takes into account the activity and the concentrationof reagent needed. The least expensive reagent tested, CTAB,is a cationic detergent that has been shownto transfectDNA into mouse L929 fibroblastcells when combinedwith DOPEat high DNA concentrations(10). Using this reagent, we found little or no transfectionof the primarycells with 5 jlg of DNA. Increasingthe DNA fourfolddid not result in increasedtransfection(datanot shown).LipofectAminehas been reportedby otherinvestigatorsto be a costeffective reagent for DNA transfectionin established lines because it gives maximal activity at very low concentrations(8). Our data indicates that LipofectAmineis not effectivein primaryhepatocytes.


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Concentration of DOTAP in gtg FIG.2. Optimumconcentrationof DOTAPrequiredto effectivelytransfect 5 jlg of plasmidDNA.The experimentwas performedon duplicateplates fromtwo differenthepatocytepreparations.Results are an averageof these two runs, n = 4. Luciferaseactivity is reportedas in Fig. 1.

DOTAP liposomes are currentlythe most economical to prepare when comparedto othercommerciallyavailablelipids. Table 1 outlinesthe relative cost per plate of the reagentstested as comparedto DOTAP.Commercially preparedLipofectinreagent costs approximately140 times more per plate thanthe DOTAPreagentpreparedfromthe nativelipid. It is forthese reasons that we have concentratedon DOTAPas a cost-effectivetransfectionagent. Fig. 2 shows the optimumconcentrationfor DOTAPrequiredto effectively transfectprimaryhepatocyteswith 5 jlg of our reportergene constructencoding luciferase.Hepatocyteswere transfectedwith vortexedDOTAPvesicles at either 6 h or 18 h after plating to determinethe optimaltransfection time. The possibilityexists that the ability of cationic lipids to transfectnucleic acids into cells may be greatlyaltered once these cells fully adhereto tissue culture dishes. However,we observedno significantdifferencein luciferase activitywhen cells were transfectedat 6 h or 18 h afterplating.Both time points displayedluciferasevalues that averagedapproximately500,000 protein. CPM/jLg Cells were incubatedwith the DNA-lipidcomplexfor differentamountsof time in orderto determinethe optimallength of time requiredfor uptake of the vesicles. Fig. 3 shows that 2 h is sufficienttime for incubationwith the DOTAPvesicles. Experiments repeated using DOTAP vesicles prepared by sonication droppedthe transfectionrateby 50%. This findingagreeswithpreviousstudies, which used a varietyof otherlipids, such as DOTMA,that betterresults are achievedby vortexingthe lipids ratherthansonicatingthem(2). Vortexing is believed to producelarge, multilamellarvesicles (2). Sonicationappears to break up the vesicles makingthem small, unilamellarvesicles (1). The DOTAPpreparationsthat were vortexedgave the highest level of luciferase reportergene expression. Morphologicalstudies.Lipofectinand DOTAParebothcommerciallyavailable, and appear to have similar transfectionrates; however,DOTAPwas specifically designed to be readily metabolizedby cells in culture and has been shown to readily bind with the cells once complexed to DNA (12). Therefore,we decided to examineLipofectinand DOTAPmediatedtransfection by electronmicroscopy.The mammalianliver consists of severaldifferent cell types the main types being hepatocytes, endothelial, Kupffer,and Ito cells (5). Hepatocyteplasma membraneassociation of the Lipofectin and DOTAPtransfectionagents had morphologicallysimilar ultrastructuralprofiles. The vesicles were bound by osmiphilic lipid bilayer membranesthat contained clumped DNA-like materialstained characteristicallyby uranyl acetate. Qualitatively,the DOTAPvesicles were usually found in clusters (Fig. 4 A) with Lipofectinvesicles more commonlyseen as single vesicles (Fig. 4 B). Distinct hepatocellularmembranemodificationswere observed

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Time in Hours FIG.3. The effect of transfectiontime on reportergene expression. Hepatocytes were transfectedwith 35 jlg of DOTAPcomplexed with 5 jlg of plasmid DNA, 18 h afterplating.At the indicatedtime points, the cells were washed of transfectionmedium, and fresh serum-freeWaymouth'smedium was added. The cells were harvested36 h aftertransfection.The experiment was performedwith two different hepatocyte preparations.Each condition included duplicate or triplicateplates. Results are an average of these two runs, n = 5. Errorbarsrepresentthe standarderrorof the mean. Luciferase activity is reportedas in Figs. 1 and 2. morphologically,demonstratingconcertedcell/vesicle association(Fig. 4 B). These membraneevents are consistent with early endocytosis described by Zhou and Huang (17). Endosomeswere readily observedin DOTAP-treated cells with both single and clustered vesicles (Fig. 4 C and D). Kupffercells wereobservedwithmultiplelargephagolysosomescontainingvesicles similar to those found in contactwith the surface of the cell. Fig. 4 A and B showsthat Lipofectinand DOTAPvesicles are very similar in size, approximately1.0 pm in diameter.This data suggests that the size of the vesicles may be an importantfactorin how the DNA-lipid complexes form,and the amountof DNA that becomes entrappedwithin the vesicles. There are currentlytwo general theories to explain the mechanismof cationic lipid mediatedDNA transfection.In the first,a single vesicle containing DNA is taken up by endocytosisand then the DNA escapes into the cytoplasm either from the endosome or the secondary lysosome. The second general theoryis based on the calculationof the ratio of chargeson DNA to charges in the cationic liposome. This calculation suggests that four liposomes of Lipofectinbind one DNA plasmid and that this complex then fuses with the plasma membrane(1). Ultrastructuraldata of Zhou and Huang (17) using cationic liposome-containinglipopolylysine (LPLL)appears to confirmthe multiple vesicle nature of the complex. This data along with others (7,10) indicatedthat the liposomesdo not fuse with the plasmamembrane,but that clusters of vesicles contactthe surfaceof the cell and destabilizethe plasma membranein that regionsufficientlyenoughto gain entrydirectlyor through endosomemembranes.The DNA is then freed withinthe cytoplasm.Vesicle clusters could be seen within the cytoplasm of hepatocytes treated with DOTAP(Fig. 4 C and D). This clustering was not observed in Lipofectintreatedcells. DOTAPvesicles also appearto have a largeramountof DNAlike materialinternally(Fig. 4 A and B). Primarycells in culture can provide an excellent model for studyingthe regulationof gene expressionbecause their metabolismis often closer to that of the cells in vivo. The continued study of the effectiveness of reagents to mediate transfectionof primarycells is thereforeimportantand may lead to better and more efficient reagentsfor use in transfectingDNA into cells. ACKNOWLEDGMENTS This research was supportedin part by grants from NIAAA (AA0672806), NIH (DK43917-01A1), and an NSF and WMUMatchingFunds Instrument Award(BIR-9120140).


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FIG.4. Electronmicrographsof transfectedprimaryrat hepatocytes.Primaryhepatocyteswere incubatedwith either DOTAP-DNA complexes or Lipofectin-DNAcomplexes under optimal conditions for 2 h after which they were preparedfor thin section electron microscopy.A, Clustersof DOTAPvesicles showingthe osmiphiliclipid bilayermembranesthat contain DNA-like materialcharacteristically stainedby uranylacetate.B, Lipofectinvesicle demonstratingconcertedcell/vesicle associationwiththe cell membrane.C, DOTAP vesicle found inside an endosome within a hepatocyte.D, Clustersof DOTAPvesicles found internalizedwithin an endosome. Bars represent0.5 gtm.

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3. Felgner,P. L.; Ringold,G. M. Cationicliposome-mediatedtransfection. Nature337:387-388; 1989. 4. Fulton,R.; Van Ness, B. Luminescentreportergene assays for luciferase and 3-galactosidaseusing a liquid scintillation counter. Biotechniques 14:762-763; 1993. 5. Gabellec, M. M.; Steffan,A. M.; Dodeur,M., et al. Membranelipids of hepatocytes,Kupffercells, and endothelialcells. Biochem.Biophys. Res. Commun.113:845-852; 1983.


DNA TRANSFECTIONINTO PRIMARY CELLS 6. Jarnagin,W. R.; Debs, R. J.; Wang, S., et al. Cationic lipid-mediated transfectionof liver cells in primaryculture. Nucleic Acids Res. 20:4205-4211; 1992. 7. Leventis, R.; Silvius, J. R. Interactionsof mammaliancells with lipid dispersions containing nove metabolizable cationic amphiles. Biochim.Biophys. Acta. 1023:124-132; 1989. 8. Lucas,J. M.;Lo, K.;Wilkie, N. M., et al. Efficienttransfectionof NIH3T3 and P19 cells with LipofectAMINEreagent.Focus 16:87-89; 1994. 9. Midoux,P.; Mendes, C.; Legrand,A., et al. Specific gene transfermediated by lactosylated poly-L-lysine into hepatomacells. Nucleic Acids Res. 21:871-878; 1993. 10. Pinnaduwage,P.; Schmitt,L.; Huang,L. Use of a quaternaryammonium detergentin liposome mediated DNA transfectionof mouse L-cells. Biochim.Biophys. Acta 985:33-37; 1989. 11. Rank,K. B.; Harris,P. K.; Ginsberg,L. C., et al. Isolationand sequence of a rat glucose-6-phosphatedehydrogenasepromoter.Biochim.Biophys. Acta 1217:90-92; 1994.

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12. Reifenrath,B.; Abken, H. DNA transfectionof human lymphoid cell lines using DOTAP.Biochemica. 11:12; 1994. 13. Rippe, R. A.; Brenner,D. A.; Leffert,H. L. DNA-mediatedgene transfer into adult rathepatocytesin primaryculture.Mol.Cell. Biol. 10:689695; 1990. 14. Rose, J. K.; Buonocore,L.;Whitt,M. A. A new cationicliposomereagent mediating nearly quantitativetransfectionof animal cells. Biotechniques 10:520-525; 1991. 15. Sambrook,J.; Fritsch, E. M.; Maniatis,T. In: Nolan, C. ed. Molecular cloning: a laboratorymanual, 2nd ed. Vol. 1. Cold Spring Harbor, NY: Cold SpringHarborLaboratoryPress; 1989:1.25-1.28. 16. Stapleton,S. R.; Stevens, G. J.; Teel, J. F., et al. Effects of acetaldehyde on glucose-6-phosphatedehydrogenaseactivityand mRNAlevels in primaryrat hepatocytesin culture.Biochimie 75:971-976; 1993. 17. Zhou, X.; Huang, L. DNA transfectionmediated by cationic liposomes containinglipopolylysine:characterizationand mechanismof action. Biochim. Biophys.Acta 1189:195-203; 1994.
