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Investigation of factors responsible for cell line cytoplasmic expression differences Finn, Jonathan D; Wong, Tabitha; Cullis, Pieter R May 11, 2005

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ralssBioMed CentBMC Molecular BiologyOpen AcceResearch articleInvestigation of factors responsible for cell line cytoplasmic expression differencesJonathan D Finn*1, Tabitha Wong1 and Pieter R Cullis1,2Address: 1Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, B.C., Canada and 2Inex Pharmaceuticals Corp., Burnaby, B.C., CanadaEmail: Jonathan D Finn* - jdfinn@interchange.ubc.ca; Tabitha Wong - twong@interchange.ubc.ca; Pieter R Cullis - pieterc@interchange.ubc.ca* Corresponding author    AbstractBackground: Previous work has described a novel cytoplasmic expression system that results ina 20-fold increase in the levels of gene expression over a standard CMV-based nuclear expressionsystem, as compared with a 2–3 fold increase seen with previous similar systems. While thisincrease was seen with BHK and Neuro-2a cells, further studies revealed that some cell lines, suchas COS-7, demonstrated relatively poor levels of cytoplasmic expression. The objective of thisstudy was to determine what factors were responsible for the different expression levels betweenBHK (a high expressing cell line) and COS-7 (a low expressing cell line).Results: The main findings of this work are that the individual elements of the cytoplasmicexpression system (such as the T7 RNAP gene and Internal Ribosome Entry Sequence) arefunctioning similarly in both cell types. Both cell types were found to have the same amount ofcytosolic nuclease activity, and that the cells appeared to have differences in the intra-cellularprocessing of DNA -cationic lipid complexes.Conclusion: After exploring many factors, it was found that differences in the intra-cellularprocessing of the DNA-cationic lipid complex was the most probable factor responsible for thedifference in cytoplasmic gene expression.BackgroundPrevious work has described a novel cytoplasmic expres-sion system that results in a 20-fold increase in the levelsof gene expression over a standard CMV-based nuclearexpression system [1], as compared with a 2–3 foldincrease seen with previous similar systems [2]. While thisincrease was seen with BHK and Neuro-2a cells, furtherstudies revealed that some cell lines, such as COS-7, dem-onstrated relatively poor levels of cytoplasmic expression.factor with respect to the cytosolic half-life of plasmidbased vectors. These nucleases could be important toresearchers using plasmid based delivery systems, espe-cially cytoplasmic gene expression systems.Internal Ribosome Entry Sequences (IRES) are elementsthat have been shown to drive expression of the secondgene in a bi-cistronic mRNA transcript, as well as increasethe translation of un-capped transcripts. IRES elementswere first isolated from viral genomes, such as the enceph-Published: 11 May 2005BMC Molecular Biology 2005, 6:11 doi:10.1186/1471-2199-6-11Received: 05 January 2005Accepted: 11 May 2005This article is available from: http://www.biomedcentral.com/1471-2199/6/11© 2005 Finn et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Page 1 of 9(page number not for citation purposes)Cytosolic nucleases have been discovered and character-ized [3,4]. These proteins have found to be an importantalomyocarditis virus (EMCV), where they allow the trans-lation of viral mRNA transcripts, made in the cytoplasm,BMC Molecular Biology 2005, 6:11 http://www.biomedcentral.com/1471-2199/6/11and thus lacking the 5' cap structure essential for mRNAtranslation [5]. Various viral IRES elements, such as theEMCV, FMDV (foot and mouth disease virus), and otherpicornavirus based IRES elements share similar features.All are approx 450 bp long and share a conserved second-ary structure, as well as a pyrimidine-rich tract that starts~25 bp before the 3' end of the IRES [6-8]. The secondarystructure is hypothesized to allow for proper alignment ofribosome subunits and other co-factors necessary fortranslation.The objective of this study was to determine what factorswere responsible for the different expression levelsbetween BHK (a high expressing cell line) and COS-7 (alow expressing cell line). After investigating numerous fac-tors, including the Internal Ribosome Entry Sequence(IRES) element, T7 RNAP activity, cytosolic nuclease lev-els and intra-cellular processing, it was found that differ-ences in the intra-cellular processing of the DNA-cationiclipid complex was the most probable factor responsiblefor the difference in gene expression.ResultsAutogene transfection in COS-7 cells results in lower transgene expression than in BHK cellsIn previous studies, it was found that the enhanced dualpromoter autogene system demonstrated transgeneexpression levels that were 15–20 times higher than theequivalent nuclear control [1]. This was demonstrated forboth BHK and Neuro-2a cells. However, when the systemwas tested in the COS-7 cell line, the autogene systemresulted in levels of gene expression that were lower thanthe nuclear control (Figure 1). It had been hypothesizedthat the autogene system should produce high levels ofgene expression in any transfected cell. Possible factorsthat would affect autogene expression levels includingIRES function, T7 RNAP activity, cytosolic nuclease levels,and intra-cellular processing differences were thereforeexamined.EMCV IRES is functional in COS-7 cellsThe first parameter that was tested was whether or not theEMCV IRES was functional in COS-7 cells. In order todetermine this, mRNA was synthesized in vitro and trans-fected directly into either BHK or COS-7 cells. As isobserved in Figure 2, the pattern of gene expression is sim-ilar for both cell lines. This indicates that while the IREScontaining transcripts are not translated as efficiently ascapped transcripts [9], the EMCV IRES has the same rela-tive expression pattern in both of the cell types, indicatingthat differences in IRES function are most likely not thereason for the poor expression levels in COS-7 cells. It isinteresting to note that the while the pattern of geneWhile it is known that cell division is an important factorfor plasmid transfection, the BHK and COS-7 cells werefound to have similar doubling times (data not shown),ruling this out as a factor responsible for the low expres-sion levels in the COS-7 cells. Another reason could bebecause COS-7 cells are transfected (nucleic acid taken upand delivered into the cytoplasm or nucleus and subse-quently expressed) less efficiently than BHK cells, or theintracellular processing of the nucleic acid-lipid com-plexes are different in the two cell lines, with the BHK cellshaving a greater amount of intact nucleic acid beingreleased into the cytoplasm than the COS-7 cells. Thispossible difference in nucleic acid:cationic lipid complexprocessing could be responsible for the low levels of cyto-plasmic autogene expression. It should be noted that wehave used both BHK and COS-7 cells extensively and havedetermined that both cell lines are transfected with similarefficiency (~20%, data not shown), and that this effi-ciency is not dependent on the plasmid used.T7 RNAP is functional in COS-7 cellsThe next parameter that was examined was whether the T7RNA polymerase (that is the basis of the cytoplasmicexpression system) is active in COS-7 cells. Therefore, invitro synthesized T7 RNAP mRNA was transfected directlyinto BHK or COS-7 cells. Twenty-four hours later cellswere transfected with a luciferase reporter plasmid thatwould only express luciferase if functional T7 RNAP wasbeing produced in those cells. As can be seen in Figure 3,while the levels of luciferase expression were lower in theCOS-7 cells, these lower levels are consistent with theoverall lower expression levels also seen in Figure 2. Notethat in the absence of T7 RNAP pretreatment, no luciferasewas detected. This data demonstrates that the T7 RNAPwas indeed functional in the COS-7 cells. Therefore, theseresults indicate that the activity of the T7 RNAP gene wasmost likely not the reason for the poor expression levels.Effect of T7 RNAP mRNA pretreatment on autogene expressionSince it appeared as if all of the individual components ofthe autogene expression system were functioning in theCOS-7 cells, it was suggested that perhaps the poor expres-sion levels were due to temporal constraints. The cytoplas-mic expression system is dependent on the initial sourceof T7 RNAP coming from the nucleus via the CMV pro-moter, which is then able to 'trigger' the cytoplasmic sys-tem. Perhaps by the time the first T7 RNAP proteins wereproduced via the nuclear transcripts (at least 8–12 hourspost transfection), cytosolic nucleases had degraded thecytoplasmic plasmid, therefore leaving no substrate forthe T7 RNAP. In order to test this hypothesis, BHK orCOS-7 cells were transfected with T7 RNAP mRNA.Page 2 of 9(page number not for citation purposes)expression is similar, the absolute levels of gene expres-sion are much lower in the COS-7 cells than the BHK cells.Twenty-four hours post mRNA transfection cells weretransfected with a plasmid encoding either theBMC Molecular Biology 2005, 6:11 http://www.biomedcentral.com/1471-2199/6/11Autogene expression in COS-7 cells A) Diagrams of plasmids usedFigur  1Autogene expression in COS-7 cells A) Diagrams of plasmids used. B) Comparison of cytoplasmic (R011) and nuclear (L053) expression plasmids in COS-7 cells. COS-7 cells were transfected with 0.75 µg/well of plasmid. Equimolar amounts of plasmids were added, and the total mass of DNA per transfection was kept equal by adding an unrelated plasmid (pPUC19). Transfec-tions and luciferase assays were performed as described in Materials and Methods. Error bars indicate standard error.T7 RNAPLuciferaseKanR IntronEMC IRESEMC IRESCMVPT7PT7ColE1 OripR01110769 bpLuciferaseKanR IntronCMVColE1 OripL0535742 bpab0200400600800100012001400R011(cytoplasmic)(COS-7)L053 (nuclear)(COS-7)24 h48 h70 hfgLuciferase / µg Total ProteinPage 3 of 9(page number not for citation purposes)BMC Molecular Biology 2005, 6:11 http://www.biomedcentral.com/1471-2199/6/11cytoplasmic expression system (R011) or the nuclear con-trol (L053). As is apparent in Figure 4, the pretreatment ofthe COS-7 cells led to a 10 fold increase in autogene basedexpression that was not seen with the nuclear control. Itwas also observed that only about a 2–3 fold increase inexpression was seen with the BHK cells (data not shown).This result appeared to support the hypothesis that timingwas a major factor. If the cells were already primed withT7 RNAP protein prior to autogene expression, a substan-tial increase in gene expression was seen. These resultssuggest that certain factors affecting the amount of freean important role in autogene expression differencesbetween the BHK and COS-7 cells.BHK and COS-7 cells have similar levels of cytosolic nucleasesIn order to understand the difference between the highexpressing (BHK) and low expressing (COS-7) cell lines, itwas hypothesized that perhaps the BHK cells had lowerlevels of cytosolic nuclease, therefore more plasmid sur-vived in the cytoplasm during the 8–12 h lag periodbefore T7 RNAP was produced from the nuclear mRNA.Transfection of BHK or COS-7 cells with various mRNA speciesFigur  2Transfection of BHK or COS-7 cells with various mRNA species. Cells were transfected with 0.75 µg/well of mRNA. Transfec-tions and luciferase assays were performed as described in Materials and Methods. Error bars indicate standard error.050001000015000200002500030000350004000045000IRES-Luc Cap-IRES-LucCap-Luc Luc20 hIRES-Luc Cap-IRES-LucCap-Luc LucBHK COS-7fgLuciferase / µg Total ProteinPage 4 of 9(page number not for citation purposes)cytoplasmic plasmid (such as cytoplasmic nuclease levelsor the intracellular processing of complexes) are playingTherefore, cytoplasmic extracts of both cell lines wereobtained and subjected to nuclease assays. As can be seenBMC Molecular Biology 2005, 6:11 http://www.biomedcentral.com/1471-2199/6/11in Figure 5, it appears as if the BHK cells did not havelower levels of cytoplasmic nuclease activity than theCOS-7 cells, and may even appear to have slightly highernuclease activity. Therefore, it appears as if the levels ofcytosolic nuclease are not a major determining factor withrespect to autogene based cytoplasmic expression.Intracellular processing of DNA is different in BHK and COS-7 cellsWhile it had been determined that the low levels ofautogene expression in COS-7 cells was not due toincreased levels of cytosolic nucleases, it could occur dueto differences in the intracellular processing of DNA-lipidcomplexes between the two cell lines. In order to investi-gate this, plasmid DNA was labeled with a fluorescentlabel, transfected into both BHK or COS-7 cells, and thenflorescence microscopy was performed at various timepoints. As can be seen in Figure 6, while there appears tobe a similar level of plasmid taken up into both cell linesat early time points (24 h), at later time points (48 h), theBHK cells seem to have accumulated a significant amountof plasmid in punctate, perinuclear structures, while theCOS-7 cells have lost a significant amount of their fluores-cence. Thus, it appears as if the BHK cells contain a poolof plasmid DNA for a longer period than the COS-7 cells.This perinuclear pattern seen with the BHK cell is charac-teristic of DNA-lipid complexes, and previous work [10]has demonstrated that these structures are not associatedwith lysosomes, and appear to be large structures, presum-ably formed when multiple endosomes have fused intoT7 RNAP is functional in COS-7 cellsFigure 3T7 RNAP is functional in COS-7 cells. Transfection of BHK or COS-7 cells with 0.75 µg of capped T7-RNAP mRNA for 24 hours, followed by a transfection with 0.75 µg of a luci-ferase reporter construct (L059) mRNA species. Cells were harvested 20 h post plasmid transfection. Transfections and luciferase assays were performed as described in Materials and Methods. Error bars indicate standard error. Note that in the absence of T7 RNAP (no pre-transfection) no luci-ferase was detected.Pretreatment of COS-7 cells with T7 RNAP mRNAFigure 4Pretreatment of COS-7 cells with T7 RNAP mRNA. Trans-fection of COS-7 cells with 0.75 µg of capped T7-RNAP mRNA for 24 hours, followed by a transfection with 0.75 µg of R011 (cytoplasmic) or L053 (nuclear) expression plasmids. Cells were harvested 24 h post plasmid transfection. Trans-fections and luciferase assays were performed as described in Materials and Methods. Error bars indicate standard error050000100000150000200000250000300000350000Cap-RNAP +L059 (BHK)Cap-RNAP + L059 (COS-7)20 hfgLuciferase / µg Total Protein020406080100120140160R0110 ugmRNAR0110.25 ugmRNAR0110.5 ugmRNAR0111.0 ugmRNA24 hL0530 ugmRNAL0530.25 ugmRNAL0530.5 ugmRNAL0531.0 ugmRNApg Luciferase / µg Total ProteinCytosolic nuclease assays of BHK and COS-7 cytoplasmic extractsFigure 5Cytosolic nuclease assays of BHK and COS-7 cytoplasmic extracts. 200 ng/µg plasmid DNA was incubated and aliquots were taken at time points indicated. No significant difference in nuclease activity between the BHK and COS-7 cytoplasmic extracts was observed.0204060801001200 2 4 6 8 10 12Time (min)BHKCOS-7Percent of supercoiled plasmid remaining (%)Page 5 of 9(page number not for citation purposes)larger vesicles. These studies clearly show that there is adifference in the intracellular processing of the DNA-lipidBMC Molecular Biology 2005, 6:11 http://www.biomedcentral.com/1471-2199/6/11Intracellular processing of complexesFigur  6Intracellular processing of complexes. Transfection of BHK or COS-7 cells with FITC labeled R011. Cells were transfected with 0.75 µg of FITC-R011. Cells were fixed at time points indicated and subjected to phase contrast and fluorescence microscopy.Phase contrast FITC-Plasmid24 h48 hBHKCOS-7BHKCOS-7Page 6 of 9(page number not for citation purposes)BMC Molecular Biology 2005, 6:11 http://www.biomedcentral.com/1471-2199/6/11complexes between the BHK and COS-7 cells, and this ismost likely the reason behind the lower levels of cytoplas-mic gene expression seen with the COS-7 cells.DiscussionWhile previous work from our lab demonstrated a 15–20fold increase in levels of gene expression using anautogene based, cytoplasmic expression system [1] inBHK and Neuro-2a cell lines, recent studies showed thatthe levels of cytoplasmic expression were significantlylower in some other cell lines, such as COS-7. In thepresent work, an attempt is made to determine what thereason for the difference was. Here various factors are dis-cussed, including the activity of the various componentsof the autogene system in BHK and COS-7 cells, the find-ing that pretreating the COS-7 cells with T7 RNAP led toan increase in expression, and that the differences inexpression between the two cell lines is most likely due todifferences in intracellular processing of the DNA-lipidcomplexes.Other studies have shown that IRES function is dependenton trans-acting cellular factors [11]. The pyrimidine tractbinding protein (PTB) binds to the EMCV IRES [12], andfunctions by maintaining the IRES secondary structure,thereby facilitating translation through the IRES [13]. TheLa antigen has also been demonstrated to bind to the IRES[11,14]. There is also evidence for other proteins bindingto various regions of IRES elements [15] that have beenreported to have an effect on the IRES function. Therefore,it was thought that perhaps one of the differences betweenthe BHK and COS-7 cells was the presence and/or abun-dance of various trans-acting cellular factors. However,transfection with various mRNA transcripts showed anidentical pattern to that of the BHK cells, indicating thatthe EMCV IRES was functioning similarly in both of thecell lines, ruling out the IRES as the reason for the expres-sion level differences.It was then suggested that perhaps the T7 RNAP proteinwas not active in the COS-7 cells. This would also lead topoor expression levels. Transfecting the cells with cappedmRNA encoding T7 RNAP, and then following 24 h laterwith a plasmid that will only express luciferase in the pres-ence of T7 RNAP demonstrated that this was not the case.Even though the levels of expression were much lowerthan in BHK cells, a substantial amount of protein wasexpressed. It appears as if the COS-7 cells in general maynot be very amenable to mRNA transfection, as both thetransfections with the luciferase encoding mRNAtranscripts (Figure 2) as well as the T7 RNAP encodingtranscripts (Figure 3) gave much lower expression levels inthe COS-7 cells than the BHK cells. This may be indicativenucleic acid-lipid complex, allowing for less cytoplasmicdelivery of mRNA.As it appeared that the individual components of theautogene system were functioning, it was thought thatperhaps the difference was due to a matter of timing. It isknown that it takes at least 8–12 h post transfection inorder to detect any expression from the nucleus, withexpression usually peaking at around 24–48 h post-trans-fection. Perhaps in this time the majority of cytoplasmicplasmid in the COS-7 cells was degraded by cytosolicnucleases. By priming the cells with T7 RNAP (via pre-transfection of cells with T7 RNAP mRNA) prior toautogene transfection, a 10 fold increase in autogeneexpression was observed, as opposed to about a 2 foldincrease with the nuclear control (most likely due to non-specific effects associated with increased lipid or nucleicacid delivery). Thus it was clear that the lag time betweentransfection and T7 RNAP expression from the nucleuswas playing a role in the poor expression levels in COS-7cells.In order to further understand this phenomenon, it wasproposed that the COS-7 cells had higher levels of acytosolic nuclease, and this was degrading the cytoplasmicplasmid, decreasing the amount of intact plasmid left forcytoplasmic expression. However, nuclease assays oncytoplasmic extracts from BHK or COS-7 cells showedthat in fact, the COS-7 cytoplasmic extracts had less nucle-ase activity than the BHK cells. Thus, increased levels ofcytosolic nucleases in the COS-7 cells was not the reasonfor the decreased expression.The observation that the mRNA transfections gave muchlower levels of expression in COS-7 cells than BHK cellshinted at the fact that perhaps there were differences in theintracellular processing of the DNA-lipid complexes, withthe COS-7 cells allowing for less free plasmid to bereleased into the cytoplasm.By labeling the plasmid with a fluorescent label (FITC)and looking at the internal distribution of plasmid inboth BHK and COS-7 cells over time, it was determinedthat there was a qualitative difference in not only thelocalization of the labeled plasmid, but also in theamount of plasmid. It appears as if in the COS-7 cells, theplasmids are degraded more quickly, shown by the lessintense overall fluorescence as well as the diffuse greenstaining throughout the cell, indicative of plasmid degra-dation. This could be due to either a greater number ofcomplexes being transported to lysosomes in COS-7 cells,or that the complexes are being released from the endo-somes faster, and therefore being degraded by cytosolicPage 7 of 9(page number not for citation purposes)of some difference in intracellular processing of the nucleases before the cytoplasmic expression system can betriggered. In contrast, the BHK cells contained larger poolsBMC Molecular Biology 2005, 6:11 http://www.biomedcentral.com/1471-2199/6/11of plasmid DNA in discrete, non-lysosomal, perinuclearstructures, even after 48 h. It is hypothesized that perhapsplasmid is being released into the cytoplasm over time,thereby allowing more free cytoplasmic plasmid to beavailable for cytoplasmic transcription at later timepoints.ConclusionIn conclusion, it has been demonstrated that cell line dif-ferences in cytoplasmic expression (when compared tonuclear expression) are most likely due to differences inthe intra-cellular processing of the nucleic acid-cationiclipid complexes, as opposed to differences in how theindividual components (IRES, RNAP, etc.) function.MethodsPlasmidsPlasmid R011 is a bi-cistronic plasmid consisting of abasic autogene cassette (containing the T7, T3 and SP6RNAP promoters) driven by a CMV promoter and intron,as well as a downstream Photinus pyralis luciferase reportergene cassette (for construction details see Finn et al,2004). L053 consists of the CMV promoter (with intron)from NGVL3 and the Photinus pyralis luciferase gene. L059consists of a pTRI-Amp (Ambion) backbone with EMCVIRES, Photinus pyralis luciferase and beta-globin poly-ade-nylation site derived from EMC-Luc, a gift from Dr. JonWolff (Waisman Center, Wisconsin). PT7-Luc (Promega)consists of the Photinus pyralis luciferase gene driven by aT7 RNAP promoter. L080 consists of PT7-Luc with theLuciferase gene driven by both the T7 and the SP6promoter.mRNA synthesismRNA synthesis was performed using a MEGAscript highyield transcription kit (Ambion). For all transcripts usedfor in vitro transfection, plasmid template (L059 for IRES-Luc and Cap-IRES-Luc mRNA; L080 for Cap-Luc mRNA;PT7-Luc for Luc mRNA) was linearized using EcoR 1 and1 µg of each plasmid was used as per manufacturers pro-tocols. mRNA was recovered by the LiCl procedure as out-lined in the manufacturer's protocol. After LiClprecipitation, the RNA pellet was washed 3 × with 70%ethanol to completely remove any unincorporated NTPs.TransfectionsLipoplexes were formed by mixing plasmid DNA withlarge unilamellar vesicles (LUVs) composed of equimolaramounts of DOPE:DODAC (50:50) on ice and incubatedfor 20 min prior to use. All transfections were performedat a cationic lipid to plasmid DNA charge ratio of 3:1.Lipoplexes were diluted with serum-containing mediabefore addition directly to cell medium. BHK or COS-7fections. Equimolar transfections using experimental plas-mids of different sizes were achieved through the additionof an empty vector (pPUC19) to normalize the total massof DNA in each transfection. For mRNA transfections,TransMessenger reagent (Qiagen) was used to transfectdiffering amounts of various mRNA transcripts into BHKcells (30,000 cells per well in 24-well plates). An RNA toTransMessenger reagent ratio of 1:4 was used for all trans-fections. Transfections were performed as per manufac-turer's instructions. Three hours post transfection themedium was removed and replaced with fresh serum con-taining medium. Cells were harvested and subjected toluciferase assays at time points indicated. For mRNA pre-treatment studies, cells were transfected with mRNA asdescribed above, followed by a DNA transfection 24 hlater. All transfections were performed in triplicate. Data ispresented as mean values +/- standard error.Luciferase and BCA assaysCells were washed twice with 1 mL PBS followed by theaddition of 0.2 mL lysis buffer (PBS with 0.1 % Triton X-100) before being stored at -70°C. Cells were thawed and5–20 µl of sample were assayed in duplicate on a 96-wellplate. Samples were assayed using a Berthhold CentroLB960 Microplate Luminometer and Luciferase Assay Sys-tem (Promega). Standard luciferase assays were per-formed and transfection data is reported as massquantities of luciferase protein using a standard curveobtained from serial 10-fold dilutions of a 20 mg/mLPhotinus pyralis luciferase standard (Promega). Total pro-tein was quantified using a Pierce BCA assay kit as permanufacturer's instructions.Cytoplasmic extract preparationCytoplasmic extracts were prepared using a hypotonicbuffer solution. Cells (2 million) were washed with PBS(2 × 1 mL) before a 15 min incubation on ice in 500 µlhypotonic extraction buffer (10 mM Hepes-KOH pH 7.9,1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 0.2 mM PMSF,0.6% NP-40). The samples were centrifuged at 13,000 × gfor 3 min at 4°C and the supernatant was collected andsaved as the cytoplasmic fraction. Protein levels werequantitated using a Pierce BCA assay (as described above)after exchanging the hypotonic extraction buffer with PBSusing a centrifugal filter device with a MW cut off of 3000.Nuclease assay procedureVarious amounts of cytoplasmic extract were incubatedwith plasmid DNA (50–300 ng extract/µg plasmid DNA)at 37°C. At time points indicated, aliquots were removedand flash frozen in liquid nitrogen and stored at -70°Cuntil all assays were completed. Samples were thenthawed and run on a 0.8% agarose gel and bands were vis-Page 8 of 9(page number not for citation purposes)cells were plated at 30,000 cells per well in 24-well plates.The total mass of plasmid added was identical in all trans-ualized using ethidium bromide. Nuclease activityPublish with BioMed Central   and  every scientist can read your work free of charge"BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime."Sir Paul Nurse, Cancer Research UKYour research papers will be:available free of charge to the entire biomedical communitypeer reviewed and published immediately upon acceptancecited in PubMed and archived on PubMed Central BMC Molecular Biology 2005, 6:11 http://www.biomedcentral.com/1471-2199/6/11defined by the proportion of intact supercoiled plasmidleft over time.Plasmid labelingR011 (cytoplasmic expression plasmid) was labeled usinga Label IT Tracker Fluorescein Kit from Mirus Corporationfollowing manufacturers instructions. A ratio of 1 µl LabelIT Tracker Reagent per µg of plasmid DNA was used.Transfections were performed as previously described.Fluorescence microscopyAt time points indicated, cells were washed 3 × with roomtemperature PBS before fixing for 5 min with 2% parafor-maldeyde. Cells were further washed with 2 × 1 mL PBSbefore being treated with Prolong Antifade (MolecularProbes) and mounted. Pictures were taken using a ZeissAxiovert S100 fluorescence microscope. In all cases, expo-sure times were kept constant to allow for ease of compar-ison of samples.Authors' contributionsJF completed all of the experimental design and carriedout 90% of the experimental work and wrote 80% of themanuscript. TW completed all of the tissue culture associ-ated with the work. PC has written 20% of the manuscriptand was responsible for critically revising it and giving thefinal verision approval for publicationAcknowledgementsThese studies were funded by a grant from the Canadian Institutes of Health Research (CIHR), Inex Pharmaceuticals Corp. and Protiva Biother-apeutics Inc.References1. Finn J, Lee AC, MacLachlan I, Cullis P: An enhanced autogene-based dual-promoter cytoplasmic expression system yieldsincreased gene expression. Gene Ther 2004, 11:276-283.2. Brisson M, He Y, Li S, Yang JP, Huang L: A novel T7 RNA polymer-ase autogene for efficient cytoplasmic expression of targetgenes. Gene Ther 1999, 6:263-270.3. Lechardeur D, Sohn KJ, Haardt M, Joshi PB, Monck M, Graham RW,Beatty B, Squire J, O'Brodovich H, Lukacs GL: Metabolic instabilityof plasmid DNA in the cytosol: a potential barrier to genetransfer. Gene Ther 1999, 6:482-497.4. Pollard H, Toumaniantz G, Amos JL, Avet-Loiseau H, Guihard G, BehrJP, Escande D: Ca2+-sensitive cytosolic nucleases prevent effi-cient delivery to the nucleus of injected plasmids. J Gene Med2001, 3:153-164.5. Jang SK, Pestova TV, Hellen CU, Witherell GW, Wimmer E: Cap-independent translation of picornavirus RNAs: structure andfunction of the internal ribosomal entry site. Enzyme 1990,44:292-309.6. Jackson RJ, Hunt SL, Gibbs CL, Kaminski A: Internal initiation oftranslation of picornavirus RNAs. Mol Biol Rep 1994, 19:147-159.7. Jackson RJ, Kaminski A: Internal initiation of translation ineukaryotes: the picornavirus paradigm and beyond. Rna 1995,1:985-1000.8. Agol VI: The 5'-untranslated region of picornaviral genomes.Adv Virus Res 1991, 40:103-180.9. Finn J, MacLachlan I, Cullis P: Factors limiting autogene-basedcytoplasmic expression systems. FASEB Journal 2004, in press:.11. Belsham GJ, Sonenberg N: RNA-protein interactions in regula-tion of picornavirus RNA translation. Microbiol Rev 1996,60:499-511.12. Kolupaeva VG, Hellen CU, Shatsky IN: Structural analysis of theinteraction of the pyrimidine tract-binding protein with theinternal ribosomal entry site of encephalomyocarditis virusand foot-and-mouth disease virus RNAs. Rna 1996,2:1199-1212.13. Kaminski A, Jackson RJ: The polypyrimidine tract binding pro-tein (PTB) requirement for internal initiation of translationof cardiovirus RNAs is conditional rather than absolute. Rna1998, 4:626-638.14. Meerovitch K, Svitkin YV, Lee HS, Lejbkowicz F, Kenan DJ, Chan EK,Agol VI, Keene JD, Sonenberg N: La autoantigen enhances andcorrects aberrant translation of poliovirus RNA in reticulo-cyte lysate. J Virol 1993, 67:3798-3807.15. Hoffman MA, Palmenberg AC: Revertant analysis of J-K muta-tions in the encephalomyocarditis virus internal ribosomalentry site detects an altered leader protein. J Virol 1996,70:6425-6430.yours — you keep the copyrightSubmit your manuscript here:http://www.biomedcentral.com/info/publishing_adv.aspBioMedcentralPage 9 of 9(page number not for citation purposes)10. Zabner J, Fasbender AJ, Moninger T, Poellinger KA, Welsh MJ: Cel-lular and molecular barriers to gene transfer by a cationiclipid. J Biol Chem 1995, 270:18997-19007.

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