Abstract
Introduction of genetic material into cells is an essential prerequisite for current research in molecular cell biology. Although transfection with commercially available reagents results in excellent gene expression, their high costs are obstacles to experimentation with a large number or large scales of transfection. The cationic polymer linear-polyethylenimine (MW 25,000) (PEI), one of the most cost-effective vehicles, facilitates DNA compaction by polyplex formation, which leads to efficient delivery of DNA into cells by endocytosis. However, the use of PEI is still limited because of substantial cytotoxicity and intolerable deterioration in transfection efficiency by its low stability. Here, we show that acidification of PEI is important for its transfection activity. Dissolving PEI powder in 0.2N HCl confers a long shelf-life for PEI storage at 4 and −80 °C, and the polyplex formation of plasmid DNA with PEI is optimized in lactate-buffered saline at pH 4.0. Furthermore, changing the culture medium at 8–12 h posttransfection can minimize the cytotoxicity of PEI without sacrificing the high transfection efficiency comparable to that of commercial reagents. The cost per test using acidified PEI is drastically reduced to approximately 1:10,000, compared with commercial reagents. Thus, we conclude that acidification of PEI satisfactorily accomplishes cost-effective, high-efficiency transfection.
This is a preview of subscription content, log in via an institution to check access.
References
Abdallah B, Hassan A, Benoist C, Goula D, Behr JP, Demeneix BA (1996) A powerful nonviral vector for in vivo gene transfer into the adult mammalian brain: polyethylenimine. Hum Gene Ther 7:1947–1954
Boussif O, Lezoualc’h F, Zanta MA, Mergny MD, Scherman D, Demeneix B, Behr JP (1995) A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc Natl Acad Sci USA 92:7297–7301
Brissault B, Kichler A, Guis C, Leborgne C, Danos O, Cheradame H (2003) Synthesis of linear polyethylenimine derivatives for DNA transfection. Bioconjug Chem 14:581–587. doi:10.1021/bc0200529
Clamme JP, Azoulay J, Mély Y (2003) Monitoring of the formation and dissociation of polyethylenimine/DNA complexes by two photon fluorescence correlation spectroscopy. Biophys J 84:1960–1968. doi:10.1016/S0006-3495(03)75004-8
Davis ME (2002) Non-viral gene delivery systems. Curr Opin Biotechnol 13:128–131. doi:10.1016/S0958-1669(02)00294-X
Durocher Y, Perret S, Kamen A (2002) High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells. Nucleic Acids Res 30:e9
Ehrhardt C, Schmolke M, Matzke A, Knoblauch A, Will C, Wixler V, Ludwig S (2006) Polyethylenimine, a cost-effective transfection reagent. Signal Transduction 6:179–184. doi:10.1002/sita.200500073
Ferrari S, Moro E, Pettenazzo A, Behr JP, Zacchello F, Scarpa M (1997) ExGen 500 is an efficient vector for gene delivery to lung epithelial cells in vitro and in vivo. Gene Ther 4:1100–1106
Fischer D, Bieber T, Li Y, Elsässer HP, Kissel T (1999) A novel non-viral vector for DNA delivery based on low molecular weight, branched polyethylenimine: effect of molecular weight on transfection efficiency and cytotoxicity. Pharm Res 16:1273–1279. doi:10.1023/A:1014861900478
Godbey WT, Wu K, Mikos AG (2001) Poly(ethylenimine)-mediated gene delivery affects endothelial cell function and viability. Biomaterials 22:471–480. doi:10.1016/S0142-9612(00)00203-9
Goula D, Remy JS, Erbacher P, Wasowicz M, Levi G, Abdallah B, Demeneix BA (1998) Size, diffusibility and transfection performance of linear PEI/DNA complexes in the mouse central nervous system. Gene Ther 5:712–717
Greenberg SM, Rosenthal DS, Greeley TA, Tantravahi R, Handin RI (1988) Characterization of a new megakaryocytic cell line: the Dami cell. Blood 72:1968–1977
Higashiyama Y, Takahashi A, Fukumoto Y, Nakayama Y, Yamaguchi N (2009) Induction of chromatin condensation by nuclear expression of a novel arginine-rich cationic protein genetically engineered from the enhanced green fluorescent protein. Cytotechnology 60:153–159. doi: 10.1007/s10616-009-9227-7
Hirao A, Huang XL, Suda T, Yamaguchi N (1998) Overexpression of C-terminal Src kinase homologous kinase suppresses activation of Lyn tyrosine kinase required for VLA5-mediated Dami cell spreading. J Biol Chem 273:10004–10010. doi:10.1074/jbc.273.16.10004
Ira, Mély Y, Krishnamoorthy G (2003) DNA vector polyethyleneimine affects cell pH and membrane potential: a time-resolved fluorescence microscopy study. J Fluoresc 13:339–347. doi:10.1023/A:1025381812568
Menzel H, Horstmann S, Behrens P, Bärnreuther P, Krueger I, Jahns M (2003) Chemical properties of polyamines with relevance to the biomineralization of silica. Chem Commun 2003:2994–2995. doi:10.1039/b310201g
Miyazaki J, Takaki S, Araki K, Tashiro F, Tominaga A, Takatsu K, Yamamura K (1989) Expression vector system based on the chicken beta-actin promoter directs efficient production of interleukin-5. Gene 79:269–277. doi:10.1016/0378-1119(89)90209-6
Moghimi SM, Symonds P, Murray JC, Hunter AC, Debska G, Szewczyk A (2005) A two-stage poly(ethylenimine)-mediated cytotoxicity: implications for gene transfer/therapy. Mol Ther 11:990–995. doi:10.1016/j.ymthe.2005.02.010
Nakayama Y, Yamaguchi N (2005) Multi-lobulation of the nucleus in prolonged S phase by nuclear expression of Chk tyrosine kinase. Exp Cell Res 304:570–581. doi:10.1016/j.yexcr.2004.11.027
Nakayama Y, Kawana A, Igarashi A, Yamaguchi N (2006) Involvement of the N-terminal unique domain of Chk tyrosine kinase in Chk-induced tyrosine phosphorylation in the nucleus. Exp Cell Res 312:2252–2263. doi:10.1016/j.yexcr.2006.03.021
Nakayama Y, Igarashi A, Kikuchi I, Obata Y, Fukumoto Y, Yamaguchi N (2009) Bleomycin-induced over-replication involves sustained inhibition of mitotic entry through the ATM/ATR pathway. Exp Cell Res 315:2515–2528. doi:10.1016/j.yexcr.2009.06.007
Ogris M, Steinlein P, Kursa M, Mechtler K, Kircheis R, Wagner E (1998) The size of DNA/transferring-PEI complexes is an important factor for gene expression in cultured cells. Gene Ther 5:1425–1433
Ogris M, Brunner S, Schüller S, Kircheis R, Wagner E (1999) PEGylated DNA/transferrin-PEI complexes: reduced interaction with blood components, extended circulation in blood and potential for systemic gene delivery. Gene Ther 6:595–605
Pham PL, Perret S, Doan HC, Cass B, St-Laurent G, Kamen A, Durocher Y (2003) Large-scale transient transfection of serum-free suspension-growing HEK293 EBNA1 cells: peptone additives improve cell growth and transfection efficiency. Biotechnol Bioeng 84:332–342. doi:10.1002/bit.10774
Sato I, Obata Y, Kasahara K, Nakayama Y, Fukumoto Y, Yamasaki T, Yokoyama KK, Saito T, Yamaguchi N (2009) Differential trafficking of Src, Lyn, Yes and Fyn is specified by the state of palmitoylation in the SH4 domain. J Cell Sci 122:965–975. doi:10.1242/jcs.034843
Suh J, Paik HJ, Hwang BK (1994) Ionization of poly(ethylenimine) and poly(allylamine) at various pH’s. Bioorg Chem 22:318–327. doi:10.1006/bioo.1994.1025
Takahashi A, Obata Y, Fukumoto Y, Nakayama Y, Kasahara K, Kuga T, Higashiyama Y, Saito T, Yokoyama KK, Yamaguchi N (2009) Nuclear localization of Src-family tyrosine kinases is required for growth factor-induced euchromatinization. Exp Cell Res 315:1117–1141. doi:10.1016/j.yexcr.2009.02.010
Thomas M, Lu JJ, Ge Q, Zhang C, Chen J, Klibanov AM (2005) Full deacylation of polyethylenimine dramatically boosts its gene delivery efficiency and specificity to mouse lung. Proc Natl Acad Sci USA 102:5679–5684. doi:10.1073/pnas.0502067102
Tseng WC, Tang CH, Fang TY, Su LY (2007) Trehalose enhances transgene expression mediated by DNA-PEI complexes. Biotechnol Prog 23:1297–1304. doi:10.1021/bp070224m
Wightman L, Kircheis R, Rössler V, Carotta S, Ruzicka R, Kursa M, Wagner E (2001) Different behavior of branched and linear polyethylenimine for gene delivery in vitro and in vivo. J Gene Med 3:362–372. doi:10.1002/jgm.187
Zanta MA, Boussif O, Adib A, Behr JP (1997) In vitro gene delivery to hepatocytes with galactosylated polyethylenimine. Bioconjug Chem 8:839–844. doi:10.1021/bc970098f
Acknowledgments
The hybridoma cell line 7G7B6 was obtained from The Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University. This work was supported in part by grants-in-aid for Scientific Research, Global COE Program (Global Center for Education and Research in Immune Regulation and Treatment) and Special Funds for Education and Research (Development of SPECT probes for Pharmaceutical Innovation) from the Japanese Ministry of Education, Culture, Sports, Science and Technology, and a research grant from the Suzuken Memorial Foundation. Y.O. is a G-COE Research Assistant.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Fukumoto, Y., Obata, Y., Ishibashi, K. et al. Cost-effective gene transfection by DNA compaction at pH 4.0 using acidified, long shelf-life polyethylenimine. Cytotechnology 62, 73–82 (2010). https://doi.org/10.1007/s10616-010-9259-z
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10616-010-9259-z