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• W2002161216 abstract "BioTechniquesVol. 38, No. 1 BenchmarksOpen AccessBrief heat shock increases stable integration of lipid-mediated DNA transfectionsBrian L. Pipes, Farha H. Vasanwala, Tom C. Tsang, Tong Zhang, Phoebe Luo & David T. HarrisBrian L. PipesUniversity of Arizona, Tucson, AZ, USASearch for more papers by this author, Farha H. VasanwalaUniversity of Arizona, Tucson, AZ, USASearch for more papers by this author, Tom C. TsangUniversity of Arizona, Tucson, AZ, USASearch for more papers by this author, Tong ZhangUniversity of Arizona, Tucson, AZ, USASearch for more papers by this author, Phoebe LuoUniversity of Arizona, Tucson, AZ, USASearch for more papers by this author & David T. Harris*Address correspondence to: David T. Harris, Department of Microbiology and Immunology, Bldg. #90, Main Campus, University of Arizona, Tucson, AZ 85721, USA. e-mail: E-mail Address: davidh@u.arizona.eduUniversity of Arizona, Tucson, AZ, USASearch for more papers by this authorPublished Online:30 May 2018https://doi.org/10.2144/05381BM05AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInReddit Lipid mediated gene transfer (lipofection) has been widely used to transfer genes into various cell types (1–4). Lipofection works very well in many cell lines, resulting in high transient transfection efficiencies (our observations). However, the rate of DNA integration into the genome following lipid-mediated transfection is relatively low (5) as compared to other methods, such as retroviral systems. This inefficient integration has been thought to be a major disadvantage of plasmid vectors and has limited their use in gene therapy trials.We have attempted to overcome this hurdle by achieving higher rates of stable integrants in lipid-mediated transfections through treating the transfected cells with a mild heat shock. Others have attempted to increase stable integration by methods such as γ-irradiation (5), but the required doses resulted in 90% cell death. DNA damaging agents like hydrogen peroxide have also been used to increase stable integration of plasmid DNA. Doses of hydrogen peroxide that caused a significant effect on integration also caused 90% cell death (6). Treatment of cells with glycerol (7), dimethylsulfoxide (8), choroquine (9), and cell synchronization to the late G2/M phase of the cell cycle (10) enhances transfer of DNA into the cytoplasm and subsequently, incorporation into the nucleus. Our method uses a heat treatment of 10 min at 42°C immediately following lipid transfections, which results in up to a 100% increase in stable integrants assayed as colonies resistant to G418 antibiotic. Transient transfection efficiencies, monitored by flow cytometry using the enhanced green fluorescent protein (EGFP), were also found to be increased by a brief heat treatment.A human lung carcinoma cell line (A549), human colon carcinoma cell line (SW480), human breast carcinoma cell line (MCF-7), and two murine cell lines, B-16 (melanoma cell line) and 4T1 (mammary tumor cell line), were used in the experiments. The cells were plated in 60-mm2 tissue culture dishes (BD, Franklin Lakes, NJ, USA) at a density of 0.5 × 106 cells/dish in RPMI media, supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 100 U/mL penicillin/streptomycin, and 1 mM sodium pyruvate (all from Invitrogen, Carlsbad, CA, USA). The cells were incubated overnight at 37°C in a 5% CO2 incubator. The cells were transfected with the plasmid pCMV-EGFP-1, which was created by inserting the human cytomegalovirus (CMV) promoter into the multiple cloning site of pEGFP-1 (BD Biosciences Clontech, Palo Alto, CA, USA). The neomycin gene in pCMV-EGFP-1 is under the control of the simian virus 40 (SV40) promoter. Transfections were performed using the lipid DMRIE-C (Invitrogen) at a ratio of 1:4 µg DNA:µL lipid. One microgram DNA was diluted in 500 µL reduced serum Opti-MEM® (Invitrogen), and 4 µL DMRIE-C were diluted in 500 µL Opti-MEM in another tube. The two tubes were mixed and incubated for 30–45 min at room temperature. Before the addition of the transfection mixture, the cells were washed twice in Opti-MEM. Four hours after transfection, the tissue culture dish was covered with Parafilm® and fully immersed in a water bath maintained at 42°C for 10–30 min as specified. After removal from the water bath, the outside of the plate was sterilized with 70% alcohol, and the transfection media was replaced with fresh supplemented RPMI media. Controls were handled similarly, but without any heat treatment. Cells were then incubated for 24 h at 37°C, after which they were either harvested for flow cytometry to measure transient transfection efficiencies (Figure 1) or for colony plating to enumerate stable integration. To measure stable integration, cell number was determined by trypan blue dye exclusion staining, and appropriate cell numbers were seeded in 75-cm2 flasks with RPMI containing 500 µg/mL G418 antibiotic. Fourteen days after culture, only cells that have stably integrated the plasmid formed colonies, and the colonies were counted as a measure of stable integration (Figure 2). The 14-day time point was calculated from a survival curve, where, at day 14, 100% of untransfected cells were dead in media supplemented with 500 µg/mL G418. As a confirmation that the 14-day time point resulted in selection of stably integrated colonies, Southern blot analyses of clones were performed. High-molecular weight genomic DNA was chloroform/phenol-extracted from these clones, electrophoresed, blotted, and probed with a chemiluminescently labeled full-length linearized vector probe (Pierce Biotechnology, Rockford, IL, USA). Probe signal found exclusively in high-molecular weight DNA confirmed genomic integration (Figure 2).Figure 1. Increase in transient transfection efficiencies by heat treatment.Twenty-four hours after transfection and heat treatment, cells were harvested by trypsinization, and 1×106 cells were fixed in 1% paraformaldehyde for flow cytometry. Flow cytometric analysis was performed using a FACStar™ Plus flow cytometer (BD Biosciences Immunocytometry Systems, San Jose, CA, USA). Data are presented as percent cells positive for enhanced green fluorescent protein (EGFP) florescence. Experiments were carried out multiple times with data from one set of experiments shown. Data are presented as the mean±sd. Note that the sd for the heated 4T1 cells is too small to be seen on the graph.Figure 2. Increase in stable integration by heat treatment.Results are the mean of three experiments±sd. Cells were plated at a density of 10,000 cells/T-75 cm2 in 15 mL supplemented RPMI media with 500 µg/mL G418. Fourteen days after seeding, colonies were counted by staining with Turk's crystal violet stain. The 14-day time point was determined by a survival curve. Insert shows representative Southern blot analysis data confirming stable integration of vectors. High-molecular weight DNA from stably transfected A549 cells was restricted with NotI +/- EcoRI or left uncut. These DNAs were electrophoresed, blotted, and probed with a chemiluminescent-labeled full-length-linearized vector probe. Lane 1, molecular weight markers (HindIII-λ); lane 2, linearized vector DNA; lane 3, uncut A549 genomic DNA; lane 4, NotI cut; lane 5, NotI+EcoRI cut; lane 6, untransfected A549 genomic DNA. Note that the sd for the unheated SW480 cells is zero.Results indicated that a 20%-70% increase above the 37°C values in transient transfection efficiencies were observed as compared to the unheated cells in all cell types in multiple experiments (Figure 1). Besides the 4T1 cell line, which showed a 70% increase in transient increase in EGFP expression, the other cell lines showed an increase of 20%-30% in EGFP expression, which, although modest, was a significant increase (P<0.05). The number of stable colonies obtained was also higher in cells that had been treated with heat as compared with the control cultures. In all five cell lines, there was an increase in stable integration ranging from 50%-100% increase in stable colonies over control cells at 37°C. A 10-min treatment at 42°C led to a 90% increase in the number of G418-resistant colonies for SW480 cells. For A549 cells, there was an increase of approximately 96% in the number of G418-resistant colonies, and for MCF-7 cells, there was an increase of 55% in G418-resistant colonies (Figure 2). The murine B-16 cell line showed a 108% increase, and the 4T1 cell line had a 52% increase in stable colonies. It is important to note that heat treatment of 42°C for 10 min is mild in nature with treated cells, maintaining a viability of over 90% after heat treatment as measured by trypan blue staining (data not shown).In conclusion, we report an empirically observed phenomenon of enhanced stable plasmid integration in neoplastic cells by a brief (10 min) and mild heat treatment (42°C) following DMRIE-C lipid transfections. A similar phenomenon is seen in bacterial transformations where a brief (90 s) 42°C heat treatment increases transformation efficiencies (11). We have not studied the mechanisms that affect the increased integration rates, but it is clear from our data that heat is affecting the cell at two different levels. First, the increase in GFP-positive cells suggests an increase in the number of cells that had taken up the plasmid, possibly by affecting fluidity of the cell membrane (12). Secondly, the increased stable integration rate indicates that in more cells the DNA was able to cross the nuclear membrane and integrate into the chromosome, possibly due to a change in fluidity in the nuclear membrane or a change in chromatin structure (5), thus allowing the plasmid greater access to the chromatin. This technique could have potential in vitro applications for laboratories routinely using lipid-mediated transfections. The effect of heat on other types of lipid-mediated transfections remains to be determined.AcknowledgmentsThe authors would like to thank Marc Friedman for initial work on transfection characterization and Barb Carolus for help with flow cytometry.Competing Interests StatementThe authors declare no competing interests.References1. Felgner, P.L., T.R. Gadek, M. Holm, R. Roman, H.W. Chan, M. Wenz, J.P. Northrop, G.M. Ringold, and M. Danielsen. 1987. Lipofection: a highly efficient, lipid mediated DNA—transfection procedure. Proc. Natl. Acad. Sci. USA 84:7413–7416.Crossref, Medline, CAS, Google Scholar2. Felgner, J.H., R. Kumar, C.N. Sridhar, C.J. Wheeler, Y.J. Tsai, R. Border, P. Ramsey, M. Martin, and P.L. Felgner. 1994. Enhanced gene delivery and mechanism studies with a novel series of cationic lipid formulations. J. Biol. Chem. 269:2550–2561.Crossref, Medline, CAS, Google Scholar3. Zhdanov, R.I., N.G. Kutsenko, O.V. Podobed, O.A. Buneeva, T.A. Tsvetkova, S.O. Guriev, T.P. Lavrenova, G.A. Serebrennikova, et al.. 1998. New cationic liposomes for transfection of eukaryotic cells. Dokl. Akad. Nauk. 362:557–560.Medline, CAS, Google Scholar4. Liu, D., T. Ren, and X. Gao. 2003. Cationic transfection lipids. Curr. Med. Chem. 10:1307–1315.Crossref, Medline, CAS, Google Scholar5. Stevens, C.W., M. Zeng, and G.J. Cerniglia. 1996. Ionizing radiation greatly improves gene transfer efficiency in mammalian cells. Hum. Gene Ther. Sep. 7:1727–1734.Crossref, Medline, CAS, Google Scholar6. Stevens, C.W., G.J. Cerniglia, A.R. Giandomenico, and C.J. Koch. 1998. DNA damaging agents improve stable gene transfer efficiency in mammalian cells. Radiat. Oncol. Investig. 6:1–9.Crossref, Medline, CAS, Google Scholar7. Frost, E. and J. Williams. 1978. Mapping temperature-sensitive and host-range mutations of adenovirus type 5 by marker rescue. Virology 91:39–50.Crossref, Medline, CAS, Google Scholar8. Stow, N.D. and N.M. Wilkie. 1976. 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Sci. 23:369–374.Crossref, Medline, CAS, Google ScholarFiguresReferencesRelatedDetailsCited ByGene delivery in adherent and suspension cells using the combined physical methods3 February 2022 | Cytotechnology, Vol. 74, No. 2Which one of the thermal approaches (heating DNA or cells) enhances the gene expression in mammalian cells?5 September 2021 | Biotechnology Letters, Vol. 43, No. 10In Vitro Anti-Viral Effects of Small Heat Shock Proteins 20 and 27: A Novel Therapeutic ApproachCurrent Pharmaceutical Biotechnology, Vol. 20, No. 12Modified DCs and MSCs with HPV E7 antigen and small Hsps: Which one is the most potent strategy for eradication of tumors?Molecular Immunology, Vol. 108Delivery of molecular cargoes in normal and cancer cell lines using non-viral delivery systems9 April 2018 | Biotechnology Letters, Vol. 40, No. 6Enhancing Mechanism of Gene Transfection by Heat ShockChemistry Letters, Vol. 46, No. 8 Vol. 38, No. 1 Follow us on social media for the latest updates Metrics History Received 26 September 2003 Accepted 25 August 2004 Published online 30 May 2018 Published in print January 2005 Information© 2005 Author(s)AcknowledgmentsThe authors would like to thank Marc Friedman for initial work on transfection characterization and Barb Carolus for help with flow cytometry.Competing Interests StatementThe authors declare no competing interests.PDF download" @default.
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