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Journal of Virological Methods 145 (2007) 14–21

Selection of an optimal RNA transfection reagent and comparison toelectroporation for the delivery of viral RNA

Gladys Gonzalez a, Loretta Pfannes a,1, Robert Brazas b, Rob Striker a,∗a Departments of Medicine and Medical Microbiology and Immunology, University of Wisconsin, 1300 University Avenue, Madison, WI 53706, USA

b Mirus Bio Corporation, 505 S. Rosa Road, Suite 104, Madison, WI 53719, USA

Received 19 November 2006; received in revised form 30 March 2007; accepted 26 April 2007Available online 11 June 2007

bstract

The initiation of viral RNA replication by the transfection of viral RNA is an integral tool in dissecting the life cycles, susceptibility, andathogenesis of numerous RNA viruses. Many different transfection methods deliver viral RNA into mammalian cells, including DEAE-dextrannd lipid-based reagents, but electroporation is one of the most popular methods. Unfortunately, electroporation suffers from many limitations,ncluding high cell death, serum-free transfection conditions, and requires many cells and relatively large amounts of RNA. To optimize andacilitate the introduction of viral RNAs into mammalian cells, different commercially available RNA transfection reagents were compared forheir ability to deliver yellow fever virus (YFV) and hepatitis C virus (HCV) RNA replicons into Huh7 cells. The performance of the commercial

ransfection reagents was also compared directly to electroporation. When properly optimized, certain reagents were superior to electroporation,ith much less cell death, less RNA required and increased transfection efficiency. The factors associated with high efficiency transfection, and

he advantage of being able to deliver RNA in the presence of serum are discussed.2007 Elsevier B.V. All rights reserved.

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eywords: Viral; RNA; Transfection; Electroporation; Lipid; HCV

. Introduction

RNA viruses are important causes of many different humannd animal diseases. Researchers are continually investigatingow viruses enter and exit cells, replicate and cause disease.olecular clones are critical to understanding these processes

s well as developing effective therapies and vaccines. Whileor some viruses infectious RNA can be generated in situ byelivery of cDNA and a DNA-dependent RNA polymerase, forther viruses a more physiologically relevant delivery methods preferred, or even required. One of the great breakthroughs inhe study of positive-strand RNA viruses was the discovery that

ransfection of genomic RNA synthesized by in vitro transcrip-ion of a cDNA copy of the RNA genome could initiate the viralife cycle and produce infectious virus (Kaplan et al., 1985).

∗ Corresponding author. Tel.: +1 608 263 2994; fax: +1 608 262 8418.E-mail addresses: rstriker@wisc.edu, rtsriker@wisc.edu (R. Striker).

1 Present address: Hematology Branch, NHLBI, NIH, Building 10-CRC,oom 3-5140, 10 Center Drive, Bethesda, MD 20892-1202, USA.

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166-0934/$ – see front matter © 2007 Elsevier B.V. All rights reserved.oi:10.1016/j.jviromet.2007.04.013

his technique allowed researchers to create defined mutationsithin a viral cDNA clone and then produce viruses with specificutations. This advance greatly facilitated the study of positive-

trand RNA viruses and the roles of specific viral genes and RNAegulatory sequences in viral life cycles. The production of virusia transfection of genomic RNA has been applied to the studyf many different positive strand RNA viruses, including but notimited to, poliovirus (Kaplan et al., 1985), yellow fever virusYFV) (Rice et al., 1989), dengue virus (Lai et al., 1991), andepatitis C virus (HCV) (Lindenbach et al., 2005).

Many different methods are used to deliver viral RNAo mammalian cells, including DEAE-dextran, commercialransfection reagents (lipid and/or polymer based) and elec-roporation. Researchers often use electroporation to deliveriral RNAs to mammalian cells, and we have routinely usedhis method to deliver HCV and YFV mini-replicon RNAs touh7 cells. Unfortunately, there are many disadvantages to using

lectroporation over other transfection methods, including theeed for large cell numbers, large amounts of RNA, serum-freeransfection conditions, and the high level of cell death. To facil-tate our HCV and YFV studies, we investigated the efficacy

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G. Gonzalez et al. / Journal of V

f several commercially available RNA transfection reagents.ur hypothesis was that the commercially available transfection

eagents would not be subject to the limitations of electropo-ation, and we could identify a reagent that would facilitateigh transfection efficiency with low cellular toxicity. To testur hypothesis, we evaluated three commercial RNA transfec-ion reagents: DMRIE-C Reagent (Invitrogen, Carlsbad, CA),ransMessengerTM Reagent (Qiagen Inc.-USA, Valencia, CA),nd the TransIT®-mRNA Transfection Kit (Mirus Bio Corpo-ation, Madison, WI) in viral RNA transfection assays. Whilell transfection reagents required fewer cells and less RNA thanlectroporation in both transient and stable transfection assays,he TransIT®-mRNA Transfection Kit had the broadest appli-ability in both assay conditions. In direct comparisons, twoommercially available transfection reagents had higher signalo noise ratio with much less cell death than electroporation.

. Materials and methods

.1. Cell culture

The human hepatoma cell line, Huh7 (kindly providedy Stanley Lemon) was grown in Dulbecco’s modificationf eagle’s medium (DMEM) (Mediatech, Inc., Herndon, VA)upplemented 10% fetal bovine serum (Atlanta Biologicals,awrenceville, GA) and 100 units of penicillin and 10 �g/mltreptomycin (Invitrogen) in a humidified incubator at 37 ◦C and% CO2.

.2. Viral replicons and RNA synthesis

The yellow fever virus replicons YF-R.Luc2A-RP, YF-RES-Luc (�DD), and YF-GFP2A-RP (Jones et al., 2005)kindly provided by Richard Kuhn) were derived fromACNR/FLYF, a full length cDNA clone of YFV 17D (genuslavivirus, family Flaviviridae) (Bredenbeek et al., 2003).hese plasmids were linearized with XhoI and then purifiedsing QIAEX II Kit (Qiagen Inc.-USA) before serving asemplates for the in vitro transcription reactions. The hepatitis

virus replicon Ntat2ANeo(SI) (kindly provided by Stanleyemon) (Yi et al., 2002) was derived from the HCV 1bNDNA clone (genus Hepaciviruses, family Flaviviridae)Ikeda et al., 2002). The Ntat2ANeo(SI)(GND) replicationefective mutant was created by site-directed mutagenesis ofhe Ntat2ANeo(SI) plasmid. Amino acid 318 of the NS5B geneas mutated from aspartic acid to asparagine using oligos 5′-ACGATGCTCGTGAACGGGAACGACCTTGTCGTTATC-G-3′ and 5′-CGACAAGGTCGTTCCCGTTCACGAGCATC-TGCAGT-3′. The Ntat2ANeo plasmids were linearized withbaI and purified with the QIAEX II kit before serving as

emplates for in vitro transcription. The YFV RNA repliconsere synthesized by in vitro transcription of the linearizedFV replicon plasmids using the mMESSAGE mMACHINE

P6 Kit (Ambion, Austin, TX) according to the manufacturer’secommendations. The HCV RNA replicons were in vitroranscribed using the linearized HCV replicon plasmids andhe mMESSAGE mMACHINE T7 Kit (Ambion). After RNA

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ical Methods 145 (2007) 14–21 15

ynthesis was complete, the in vitro transcription reactions werereated with 1 �l of RNase-free DNase (Ambion) at 37 ◦C for5 min to degrade the DNA templates, and the RNA was thenurified from each reaction by LiCl precipitation.

.3. Viral RNA transfections using commercial transfectioneagents

.3.1. Initial optimization transfections in 12-well platesHuh7 cells were plated in 12-well plates (2.5 × 105

ells/well) 24 h prior to transfection and were appro-imately 80% confluent at the time of transfection. This level ofonfluency is in agreement with the recommended transfectiononditions of all three-reagent protocols. Transfection of cellst confluencies below the manufacturers’ recommended rangeorrelated with lower efficiency than the results presented heredata not shown). The viral RNA replicons were transfected intohe Huh7 cells using three different RNA transfection reagents,MRIE-C, TransMessengerTM, and the TransIT-mRNA Kit.ll transfections were performed in parallel on the same day.ransfections were performed according to the manufacturer’secommended protocols. Varying the amount of RNA withinhe manufacturer’s suggested range had little or no effect onuciferase yield. For each transfection condition tested, two sep-rate complex tubes were set up. Each tube had a 3× masterix that was aliquoted into triplicate wells; there were a total of

ix samples per condition. Briefly, the DMRIE-C transfectionsere performed by first washing the cells with 1 ml of Opti-EM Reduced Serum Media (Invitrogen). Opti-MEM (0.5 ml)as then added to a sterile polystyrene tube, followed by 1,, 3, or 4 �l of DMRIE-C, and mixed gently. The viral RNA1 �g) was added to each tube, mixed and immediately addedrop wise to the appropriate well. The cells were incubatedor 6 h at 37 ◦C and 5% CO2, and then the medium contain-ng the transfection complexes was removed from each wellnd replaced with 1 ml of complete growth medium (DMEMith 10% serum). The TransMessengerTM Reagent transfectionsere performed by first diluting 2 �l of Enhancer R in 46 �l ofuffer EC-R in a sterile polystyrene tube. One microgram ofiral RNA was added to the tube and mixed. The mixture wasncubated at room temperature for 5 min. Next, 2, 4, 6, or 8 �l ofransMessengerTM Reagent was added to each tube, mixed, and

ncubated for 10 min at room temperature. While the transfectionomplexes were forming, the cells were washed with 2 ml/wellf sterile phosphate buffered saline (PBS). After removal of theBS, 450 �l of Opti-MEM was added to each well, and the trans-ection complexes were added drop wise to each well. The cellsere then incubated for 6 h at 37 ◦C and 5% CO2. The mediumas removed from each well, the cells were gently washed withBS, and then 1 ml of complete growth medium (DMEM with0% serum) was added to each well. The TransIT-mRNA Kitransfections were performed by first adding 1 �g of viral RNAo 100 �l of Opti-MEM in a sterile polystyrene tube. Immedi-

tely the mRNA Boost Reagent was added to each tube followedy the TransIT-mRNA Reagent. The following amounts of eacheagent were used based on the protocol; 0.5/0.5, 0.5/1, 0.5/1.5,/1, 1/2, 1/3 (�l of mRNA Boost Reagent/�l of TransIT-mRNA

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eagent). The tubes were incubated at room temperature formin, and the complexes were added drop wise to the cells inomplete growth media with no media change. Following trans-ection, all the transfected cells were incubated at 37 ◦C and 5%O2 for 24 h and then harvested for luciferase assays. Based on

hese assays, transfections were optimized for six-well plates byoubling all reagents, including media components, cells, RNAnd transfection reagents.

.4. Colony forming assay

Huh7 cells were plated in six-well plates (5 × 105 cells/well)4 h prior to transfection and were approximately 90% con-uent at the time of transfection. The HCV Ntat2ANeo(SI)nd Ntat2ANeo(SI)(GND) RNA replicons were transfectednto duplicate wells of Huh7 cells using optimal DMRIE-, TransMessengerTM Reagent, and the TransIT-mRNA Kitonditions found in Section 2.3.1. Twenty-four hours post-ransfection, each well of transfected cells was trypsinizednd resuspended in fresh complete growth media containing.5 mg/ml G418 (Invitrogen). Each well of trypsinized cells waslated in two 10 cm dishes in the presence of G418. One disheceived 1/10 of the cells and the other received 9/10 of the cells.he cells were grown for 1 week in 0.5 mg/ml G418 and thenhifted to 1.0 mg/ml G418 containing media for 2 additionaleeks, during which time the media was changed every 72 h.he G418 resistant colonies that developed were visualized bytaining with Crystal Violet (Sigma–Aldrich, St. Louis, MO)s previously described (Kato et al., 2005), and the number ofolonies on the 1/10 plates were determined and averaged forach set of duplicates.

.5. Viral RNA electroporation

Subconfluent Huh7 cells in a T-75 flask (4 × 106 cells) wererypsinized, washed twice with Hank’s Buffered Salt SolutionCellgro Mediatech, Herndon, VA), and resuspended in 500 �lf PBS. The cells were transferred to a 4 mm gap electropo-ation cuvette (Molecular Bio Products, San Diego, CA). Fiveicrograms of either the YF-GFP2A-RP or the YF-R.Luc2A-P RNAs was added to a cuvette and electroporated with twoulses at 100 V, 125 �, and 2300 �F using an ECM630 Elec-roporator (BTX Molecular Delivery Systems, Holliston, MA).fter electroporation, the cells were allowed to recover at room

emperature for 5 min, resuspended in 2 ml of complete growthedia, and plated in six-well plates. Cells were incubated at

7 ◦C and 5% CO2 for 24 h, and then the media was replacedith fresh complete growth media. The plates were incubated

nother 24 h at 37 ◦C at which time they were approximately0% confluent. Cells were harvested and subjected to luciferasessays or flow cytometry analyses.

.6. Luciferase assays

YF-R.Luc2A-RP, or YF-IRES-Luc (�DD) transfected Huh7ells were washed with PBS (1 ml/12-well and 2 ml/6-well).he cells were lysed by the addition of Cell Lysis Buffer

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ical Methods 145 (2007) 14–21

100 �l/12-well and 250 �l/6-well) (Promega Corp., Madison,I) to each well followed by a 15-min incubation at room

emperature. Renilla luciferase assays were performed usinghe Renilla Luciferase Assay System (Promega). Briefly, 5 �lf each cell lysate was added to 100 �l of Luciferase Assayeagent I (LARI) and measured in a DLReady TD-20/20 Turneresigns Luminometer (Turner Biosystems, Sunnyvale, CA). In

he comparison of the electroporation to the TransIT-mRNAransfection Kit, the concentration of protein in each sampleas determined using the BCATM Protein Assay Kit (Pierce,ockford, IL) according to the manufacturer’s recommenda-

ions. The amount of luciferase activity in each sample was thenormalized to the amount of protein in the sample to take intoccount the low cellular density of the electroporated cells atarvest compared to the TransIT-mRNA Kit transfected cells.

.7. Fluorescence microscopy and flow cytometry analysis

.7.1. Fluorescence microscopyThe YF-GFP2A-RP and YF-IRES-Luc (�DD) RNA trans-

ected cells were washed once with 1 ml of PBS, followed byddition of 2 ml of PBS. Both phase contrast and GFP fluorescentmages of the transfected or electroporated cells were acquiredsing an inverted fluorescence microscope with a 10× lens andigital images were collected using a Zeiss Axiocam MRC digi-al camera (Axio Cam MRm, Zeiss, Germany) and Axio-Visionoftware (Axio Cam MRm, Zeiss, Germany).

.7.2. Flow cytometry analysisHuh7 cells electroporated or transfected with the YF-GFP2A-

P or YF-IRES-Luc (�DD) RNA replicons were harvested byrypsinization and washed twice with PBS. The cells were resus-ended in 1 ml of PBS, and subjected to flow cytometry on a LSRlow Cytometer (Becton Dickinson, Franklin Lakes, NJ). TheF-IRES-Luc (�DD) transfected cells served as the negativeFP control and were used to set the gates to identify the GFPositive cells.

. Results

.1. Comparison of three different commercial RNAransfection reagents

.1.1. Transfection of the YF-R.Luc2a-RP RNA repliconTo identify a commercial transfection reagent that might

utperform electroporation, three commercially available trans-ection reagents capable of delivering large RNA molecules toammalian cells were tested. DMRIE-C, TransMessengerTM

eagent, and the TransIT-mRNA Transfection Kit were testedn parallel for their ability to transfect the yellow fever virus YF-.Luc2A-RP RNA replicon. A range of reagent concentrationsere tested based on manufacturer’s recommendations to test

he performance of each reagent and identify the optimal con-

itions for transfecting the YF-R.Luc2A-RP RNA into Huh7ells. As expected, all three transfection reagents were capablef delivering the YFV RNA replicon to Huh7 cells (Fig. 1). TheransIT-mRNA Kit was the most efficient at transfecting the

G. Gonzalez et al. / Journal of Virolog

Fig. 1. Comparison of three commercially available RNA transfection reagents.Huh7 cells in 12-well plates were transfected with 1 �g of YF-R.Luc2A-RPRNA per well in parallel using either the TransIT®-mRNA Transfection Kit,DMRIE-C, or TransMessengerTM Reagent. The amounts of each reagent testedare indicated. Each transfection condition was tested in duplicate, and eachcomplex formation tube was set up as a 3× master mix and transfected intothree wells of a 12-well plate for a total of six wells per condition. Cells werehr

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arvested 24 h post-transfection and assayed for Renilla luciferase activity. Theesults presented are the average of the six wells transfected per condition.

eplicon RNA, producing levels of Renilla luciferase that werepproximately 250% and 84% greater than the DMRIE-C andransMessengerTM Reagent transfected cells, respectively. Theptimal TransIT-mRNA Kit transfection conditions per well of

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ig. 2. Performance of the RNA transfection reagents in a colony formation assay.tat2ANeo(SI) RNA or the negative control Ntat2ANeo(SI)(GND) RNA using the Twenty-four hours post-transfection, each well of transfected cells was trypsinizedf G418 to select for cells stably replicating the Ntat2ANeo(SI) replicon. Three weeisualize the G418 resistant colonies. (a) Colony formation after transfection using erom the duplicate transfections using the three transfection reagents.

ical Methods 145 (2007) 14–21 17

12-well plate were 2 �l TransIT-mRNA Reagent, 1 �l mRNAoost Reagent, and 1 �g of replicon RNA.

.1.2. Colony forming assay using the HCV Ntat2ANeo(SI)NA replicon

Because the optimal delivery method for transient trans-ections may differ from the optimal method for stableransfections, a colony forming assay was performed in whichhe HCV Ntat2ANeo(SI) and Ntat2ANeo(SI)(GND) RNA repli-ons were transfected into Huh7 cells using the optimalransfection conditions for each transfection reagent determinedreviously (Section 3.1.1) (Fig. 1). The Ntat2ANeo(SI) repli-on replicates in transfected cells and produces G418 resistantells, while the polymerase mutant Ntat2ANeo(SI)(GND) repli-on does not replicate and served as a negative control. Afterransfecting Huh7 cells with each RNA replicon, the cells wereplit and replated in media containing G418 and allowed to growor 3 weeks. The cultures were then stained with Crystal Violet toisualize the G418 resistant colonies formed (Fig. 2a). Very fewolonies formed after transfection of the Ntat2ANeo(SI)(GND)egative control replicon, indicating that the G418 selection wasffective (Fig. 2a). Many colonies formed after transfection ofhe Ntat2ANeo(SI) RNA with the three different transfectioneagents. In this case, the TransIT-mRNA Kit produced a largerumber of colonies than the DMRIE-C and TransMessengerTM

eagents by 20% and 53%, respectively (Fig. 2b).In summary, the three commercial transfection reagents were

ffective in both transient and stable RNA transfections, buthe efficiency was both assay and reagent dependent. The

Huh7 cells in six-well plates were transfected in duplicate with 2 �g of theransIT®-mRNA Transfection Kit, DMRIE-C, or TransMessengerTM Reagent.and 1/10 of the trypsinized cells were plated in 10 cm dishes in the presenceks post-transfection, the 10 cm dishes were stained with crystal violet blue toach of the three transfection reagents. (b) Average number of colonies formed

1 irological Methods 145 (2007) 14–21

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Fig. 3. Performance comparison of electroporation to TransIT®-mRNA Kittransfected cells using a luciferase expressing replicon. Huh7 cells were eitherelectrorporated with 5 �g or transfected with 2 �g of the YF-R.Luc2a-RP RNAusing the TransIT-mRNA Transfection Kit. Twenty-four hours post-delivery, thecells were harvested, lysed and assayed for both Renilla luciferase activity andtotal protein concentration. The luciferase activities were then normalized to

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8 G. Gonzalez et al. / Journal of V

ransIT-mRNA Transfection Kit outperformed the DMRIE-and the TransMessengerTM Reagents in both sets of tests,

lthough the difference in the colony-forming assay wasarginal.

.2. Comparison of TransIT-mRNA Kit transfections tolectroporation

.2.1. YFV luciferase replicon delivery comparisonElectroporation has commonly been used successfully to

eliver viral RNA, however since many HCV replicons areot robust replicators in vitro, these experiments require opti-al viral RNA delivery, particularly for mutants with decreased

eplication capacity (data not shown). Because of this require-ent and the high performance of the transfection reagents

n Fig. 1, the TransIT-mRNA Kit was directly compared tolectroporation. Huh7 cells with 5 �g of either the YF-R.Luc2A-P RNA or the YF-IRES-Luc (�DD) were electroporatedsing optimal conditions identified previously (data not shown)Fig. 3). The YF-IRES-Luc (�DD) RNA mutant is incapablef replicating in transfected cells due to a polymerase muta-ion. This replicon is a negative control and produced minimal

uciferase activity after delivery using either method (data nothown). In parallel, Huh7 cells were transfected with the samewo viral replicons using the TransIT-mRNA Kit, but in theseransfections only 2 �g of viral RNA per well was transfected,

total protein to account for differences in sample cell number.

ig. 4. Efficiency comparison of electroporation to transfection using a GFP expressing replicon. Huh7 cells were electroporated with 5 �g or transfected usinghe TransIT-mRNA Transfection Kit, TransMessengerTM or DMRIE-C with 2 �g of the yellow fever virus YF-GFP2A-RP or the YF-IRES-Luc (�DD) (negativeontrol) RNA using optimal conditioned determined previously. Forty-eight hours post-delivery, the cells were examined by fluorescence microscopy to visualizehe GFP transfected cells (a), or quantitated by flow cytometry to determine the number of GFP positive cells in the population (b). The YF-IRES-Luc (�DD) RNAransfected cells served as the negative control to set the gates on the flow cytometer. Note the y axis differs between RNA delivery methods and denotes cell survival,he x axis denotes fluorescence intensity.

G. Gonzalez et al. / Journal of Virological Methods 145 (2007) 14–21 19

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ased on the optimal results obtained in Section 3.1.1. Due tohe high cell death associated with electroporation, the luciferasectivity in each sample was normalized to the amount of proteinn the sample, and those normalized luciferase activities wereresented. We found that the TransIT-mRNA Kit was muchore effective at delivering the YFV replicon than electro-

oration. Normalized luciferase activities were approximately0-fold higher in the TransIT-mRNA Kit transfected sampleshan the electroporated samples (Fig. 3).

.2.2. YFV GFP replicon delivery comparisonThe YFV luciferase replicon transfections (Section 3.2.1)

emonstrated the highly effective transfection of viral RNAsing the commercial transfection reagents, however there waso direct assessment of transfection efficiency. To extend theesults of these experiments, we transfected or electroporated

uh7 cells in parallel as described in Section 3.2.1 with either

he YFV YF-GFP2A-RP or the YF-IRES-Luc (�DD) RNAs.he YF-GFP2A-RP replicon was used to determine transfec-

ion efficiency (percent GFP expressing cells in the population)

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f each delivery method. Forty-eight hours post-transfection theells were examined by fluorescence microscopy to visualize theFP expressing cells (Fig. 4a). In parallel, duplicate transfected

ells were harvested and analyzed by flow cytometry to deter-ine the percentage of GFP expressing cells in the transfected

r electroporated samples. As expected, the cells transfectedr electroporated with the YF-IRES-Luc (�DD) negative con-rol RNA did not produce any GFP expressing cells (Fig. 4b).n contrast, GFP expressing cells were detected in the Huh7ells transfected or electroporated with the YF-GFP2A-RP RNAeplicon. Quantitation by flow cytometry demonstrated that theells transfected with only 2 �g of the YF-GFP2A-RP RNAsing the TransIT-mRNA Kit produced approximately 50% GFPositive cells, while the cells electroporated with five timesore viral RNA yielded approximately 1% GFP positive cells

Fig. 4b). Transfection with the other commercial transfection

eagents (using 2 �g of RNA, the same as TransIT-mRNA Kit,ut less than electroporation) with DMRIE-C yielding resultsimilar to electroporation (∼1%) and TransMessengerTM yield-ng ∼10% transfection efficiency.

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. Discussion

The delivery of in vitro transcribed RNA to cells is a cru-ial and common technique in RNA virology. This processas been the only reliable way to study HCV replication inell culture and typically has been performed by electropora-ion. Since electroporation of HCV replicons was a particularlyow efficiency process, it became clear that selection at bothhe viral and cellular levels occurred (Bartenschlager et al.,003; Blight et al., 2002). For example, multiple labs havehown only subpopulations of Huh7 cells are highly trans-ectable (Blight et al., 2002). Since electroporation results inlarge amount of cell death, cells that receive and propagate

he replicons have passed through a significant genetic bot-leneck. Examining the aberrant cell signaling in the resultantighly permissive subpopulations such as the Huh7.5 cells haseen fruitful (Sumpter et al., 2005), but this amount of cellularelection greatly increases the complexity of experimental inter-retation. The HCV field is particularly preoccupied with drugusceptibility testing of replicons, and therefore often resorts toransient assays with less host cell selective pressure (Murrayt al., 2003).

Although electroporation is capable of delivering viral RNAo mammalian cells with reproducible and interpretable resultsBredenbeek et al., 2003; Jones et al., 2005; Lohmann et al.,999), it has several undesirable features. First, electropora-ion of mammalian cells in standard sized cuvettes generallyequires ∼5 × 106 cells per electroporation, and the number ofells required for a series of electroporations quickly becomesnmanageable. Second, due to the constraints on cuvette size andlectroporation equipment available, electroporations are notasily scaled up, although 96-well, high-throughput electropora-ion plates are becoming more common. Third, electroporationenerally results in significant cell death (hence, the need forarge numbers of cells), adding to the complexity of the exper-ments and a lower yield of cells in the electroporated samples.ourth, large amounts of RNA (2–10 �g) are generally requiredor each electroporation, and the time and expense involved inreparation of large quantities of RNA can be prohibitive. Fifthnd finally, electroporations are performed in the absence oferum, which increases the complexity of the procedure andecreases cell survival.

As described here, the TransIT-mRNA Transfection Kit effi-iently transfects YFV and HCV replicons into Huh7 cells, aiver hepatoma cell line, yet it is highly likely that this trans-ection kit will also be a useful tool for the study of otheriruses and other assays. For example, packaging assays fre-uently require serial electroporations to deliver both genomicNA, and structural protein mRNAs separately, and serial deliv-ry using a transfection reagent would significantly decrease celleath compared to serial electroporations (Jones et al., 2005).he TransIT-mRNA Transfection Kit has also been used to trans-

ect RNA into other cell lines such as Vero, A549, and BHK-21data not shown). These cell lines are popular hosts cells for other

iruses and will likely make this reagent a useful tool for thoseiruses as well. One of the more interesting characteristics ofhis transfection reagent is functional in the presence of serum.

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ical Methods 145 (2007) 14–21

t has been demonstrated previously that this reagent protectsNA from RNase degradation in vitro (data not shown), and

his protection is likely critical to facilitating RNA transfectionn the presence of serum. It is possible that this trait could betilized to allow the rescue of negative strand RNA viruses. Forxample, the rescue of respiratory syncytial virus (RSV) cur-ently involves the infection of Hep-2 cells with vaccinia virushat express T7 RNA polymerase. The infected cells are thenransfected with T7 promoter driven expression plasmids forhe RSV replication/transcription proteins N, P, L, and M2-1s well as the (+) strand anti-genomic plasmid (Bridgen andlliott, 1996). Packaging the replication/transcription proteinncoding mRNAs with TransIT-mRNA Transfection Reagentn one tube and the (−) genomic RNA in a separate tube wouldheoretically prevent hybridization, decrease toxicity of the cur-ent rescue procedure, obviate the need for vaccinia virus in theescue of negative strand RNA viruses, and simplify the entireescue procedure.

The goal of this research was to determine whether a commer-ially available transfection reagent could overcome the inherentrawbacks in electroporation and replace it. As described here,he three commercially available transfection reagents were allffective in both transient and stable assays, but the TransIT-RNA Kit outperformed the other two reagents in all three

ssays. We used both enzymatic and flow cytometry assayso compare the transfection reagents to electroporation, andndependent of the assay used, the transfection reagents com-ared favorably to electroporation although the magnitude ofhe differences varied. There are many possible reasons forhese differences, but the two most likely reasons are differencesn the assays and the viral RNAs transfected. The luciferasessays detect luciferase activity in all the luciferase express-ng cells independent of the level of luciferase present in anyne cell, while the GFP flow cytometry assay only detectsFP positive cells once a threshold of GFP protein level in

he cell is achieved. Second there were also slight sequenceariations between the viral luciferase reporters and the viralFP reporters, which could alter slightly the experimentalutcomes.

When compared directly to electroporation, the transfec-ion reagents, particularly the TransIT-mRNA Kit, were moreersatile, and overcame most of the problems associated withlectroporation. There are many advantages to using the trans-ection reagents. First, transfections can be performed ondherent cells in any size culture vessel, and the number ofells required is dependent on the size of the culture vesselnd not the transfection reagent. As a result, optimal trans-ection conditions in one cell culture plate can be adjustedo different culture vessels simply by scaling the transfectiononditions based on surface area of the culture vessel. Sec-nd, transfection reagents generally required less RNA for eachransfection compared to electroporation, yet produced betterransfection results. Finally, because the cells can be trans-ected in the presence of serum (not true for DMRIE-C andransMessengerTM), the transfection procedure is greatly sim-

lified and likely leads to increased viability of the transfectedells.

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missiveness to hepatitis C virus RNA replication through a cellular RNA

G. Gonzalez et al. / Journal of V

cknowledgements

TransIT®-mRNA Transfection Kit used in this study was pro-ided as a gift by Mirus Bio Corporation. G.G. was supportedn part by a grant to R.S. by The Partnership Fund for a Healthyuture and by NIH KO8 AZ0557750 to RS and the NIH/NIAIDegional Center of Excellence for Biodefense and Emerging

nfectious Diseases Research (RC) Program. The authors wish tocknowledge membership within and support from the Region VGreat Lakes’ RCE (NIH award 1-U54-AI-057153). The authorslso wish to thank Bryan Hughes for his help with the flowytometry analysis.

eferences

artenschlager, R., Kaul, A., Sparacio, S., 2003. Replication of the hepatitis Cvirus in cell culture. Antiviral Res. 60, 91–102.

light, K.J., McKeating, J.A., Rice, C.M., 2002. Highly permissive cell linesfor subgenomic and genomic hepatitis C virus RNA replication. J. Virol. 76,13001–13014.

redenbeek, P.J., Kooi, E.A., Lindenbach, B., Huijkman, N., Rice, C.M., Spaan,W.J., 2003. A stable full-length yellow fever virus cDNA clone and the roleof conserved RNA elements in Flavivirus replication. J. Gen. Virol. 84,1261–1268.

ridgen, A., Elliott, R.M., 1996. Rescue of a segmented negative-strand RNAvirus entirely from cloned complementary DNAs. Proc. Natl. Acad. Sci.

U.S.A. 93, 15400–15404.

keda, M., Yi, M., Li, K., Lemon, S.M., 2002. Selectable subgenomic andgenome-length dicistronic RNAs derived from an infectious molecular cloneof the HCV-N strain of hepatitis C virus replicate efficiently in cultured Huh7cells. J. Virol. 76, 2997–3006.

Y

ical Methods 145 (2007) 14–21 21

ones, C.T., Patkar, C.G., Kuhn, R.J., 2005. Construction and applications ofyellow fever virus replicons. Virology 331, 247–259.

aplan, G., Lubinski, J., Dasgupta, A., Racaniello, V.R., 1985. In vitro synthe-sis of infectious poliovirus RNA. Proc. Natl. Acad. Sci. U.S.A. 82, 8424–8428.

ato, N., Nakamura, T., Dansako, H., Namba, K., Abe, K., Nozaki, A., Naka,K., Ikeda, M., Shimotohno, K., 2005. Genetic variation and dynamics ofhepatitis C virus replicons in long-term cell culture. J. Gen. Virol. 86, 645–656.

ai, C.J., Zhao, B.T., Hori, H., Bray, M., 1991. Infectious RNA transcribed fromstably cloned full-length cDNA of dengue type 4 virus. Proc. Natl. Acad.Sci. U.S.A. 88, 5139–5143.

indenbach, B.D., Evans, M.J., Syder, A.J., Wolk, B., Tellinghuisen, T.L., Liu,C.C., Maruyama, T., Hynes, R.O., Burton, D.R., McKeating, J.A., Rice,C.M., 2005. Complete replication of hepatitis C virus in cell culture. Science309, 623–626.

ohmann, V., Korner, F., Koch, J., Herian, U., Theilmann, L., Bartenschlager,R., 1999. Replication of subgenomic hepatitis C virus RNAs in a hepatomacell line. Science 285, 110–113.

urray, E.M., Grobler, J.A., Markel, E.J., Pagnoni, M.F., Paonessa, G., Simon,A.J., Flores, O.A., 2003. Persistent replication of hepatitis C virus repli-cons expressing the beta-lactamase reporter in subpopulations of highlypermissive Huh7 cells. J. Virol. 77, 2928–2935.

ice, C.M., Grakoui, A., Galler, R., Chambers, T.J., 1989. Transcription ofinfectious yellow fever RNA from full-length cDNA templates produced byin vitro ligation. New Biol. 1, 285–296.

umpter Jr., R., Loo, Y.M., Foy, E., Li, K., Yoneyama, M., Fujita, T., Lemon,S.M., Gale Jr., M., 2005. Regulating intracellular antiviral defense and per-

helicase, RIG-I. J. Virol. 79, 2689–2699.i, M., Bodola, F., Lemon, S.M., 2002. Subgenomic hepatitis C virus replicons

inducing expression of a secreted enzymatic reporter protein. Virology 304,197–210.