Abstract
Transient gene expression has evolved into an attractive technology for the rapid production of milligram to gram amounts of recombinant proteins. This review describes the different methods for introducing foreign DNA into suitable mammalian cells with either viral or non-viral vectors. Particular emphasis is given to non-viral transient transfection which represents meanwhile the most prominent variant due to recent progress in the resulting protein productivity. Non-viral transient transfection protocols are always based on the use of specific transfection reagents or the application of an electroporation device. The corresponding methods are compared with regard to their scale-up potential, also in consideration of potential production costs. The underlying cellular pathways of plasmid DNA incorporation, cytoplasmic release and translocation into the nucleus are important details to understand the transfection principle and further improve the technology. Problems associated with the application of transient gene expression at a larger scale are also addressed. In particular, the requirement of different cell culture media conditions for plasmid DNA complex preparation (if necessary), the transfection process itself and a high titer recombinant production need to be harmonized. Strategies to improve recombinant protein productivity by increasing the cell-specific output and/or sustaining the production phase are itemized as well. This can be accomplished by enabling cells to perform episomal plasmid replication, co-transfection with other plasmids, altering the cellular metabolism, temperature reduction, supplementation of specific production enhancers or combinations thereof. A number of examples for successful applications at pilot scale are also provided.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Al-Rubeai M (ed) (2000) Cell engineering, vol 2, Transient expression. Kluwer, Dordrecht
Ames RS, Kost TA, Condreay JP (2007) BacMam technology and its application to drug discovery. Expert Opin Drug Discov 2:1669–1681
Backliwal G, Hildinger M, Hasija V, Wurm FM (2008a) High-density transfection with HEK-293 cells allows doubling of transient titers and removes need for a priori DNA complex formation with PEI. Biotechnol Bioeng 99:721–727
Backliwal G, Hildinger M, Kuettel I, Delegrange F, Hacker DL, Wurm FM (2008b) Valproic acid: a viable alternative to sodium butyrate for enhancing protein expression in mammalian cell cultures. Biotechnol Bioeng 101:182–189
Backliwal G, Hildinger M, Chenuet S, De Jesus M, Wurm FM (2008c) Coexpression of acidic fibroblast growth factor enhances specific productivity and antibody titers in transiently transfected HEK293 cells. N Biotechnol 25:162–166
Backliwal G, Hildinger M, Chenuet S, Wulhfard S, De Jesus M, Wurm FM (2008d) Rational vector design and multi-pathway modulation of HEK 293E cells yield recombinant antibody titers exceeding 1 g/l by transient transfection under serum-free conditions. Nucleic Acids Res 36:e96
Balasuriya UBR, Heidner HW, Hedges JF, Williams JC, Davis NL, Johnston RE, MacLachlan NJ (2000) Expression of the two major envelope proteins of equine arteritis virus as a heterodimer is necessary for induction of neutralizing antibodies in mice immunized with recombinant Venezuelan Equine Encephalitis Virus replicon particles. J Virol 74:10623–10630
Baldi L, Muller N, Picasso S, Jacquet R, Girard P, Thanh HP, Derow E, Wurm FM (2005) Transient gene expression in suspension HEK-293 cells: application to large-scale protein production. Biotechnol Prog 21:148–153
Barsoum J, Brown R, McKee M, Boyce FM (1997) Efficient transduction of mammalian cells by a recombinant baculovirus having the Vesicular Stomatitis Virus G glycoprotein. Hum Gene Ther 8:2011–2018
Berntzen G, Lunde E, Flobakk M, Andersen JT, Lauvrak V, Sandlie I (2005) Prolonged and increased expression of soluble Fc receptors, IgG and a TCR-Ig fusion protein by transiently transfected adherent 293E cells. J Immunol Methods 298:93–104
Bi C, Benham CJ (2004) WebSIDD: server for predicting stress-induced duplex destabilized (SIDD) sites in superhelical DNA. Bioinformatics 20:1477–1479
Blasey HD, Aubry J-P, Mazzei GJ, Bernard AR (1996) Large scale transient expression with COS cells. Cytotechnology 18:183–192
Blasey HD, Brethon B, Hovius R, Vogel H, Tairi AP, Lundström K, Rey L, Bernard AR (2000) Large scale transient 5-HT3 receptor production with the Semliki Forest Virus expression system. Cytotechnology 32:199–208
Bollin F, Dechavanne V, Chevalet L (2011) Design of experiment in CHO and HEK transient transfection condition optimization. Protein Expr Purif 78:61–68
Boussif O, Lezoualc’h F, Zanta MA, Mergny MD, Scherman D, Demeneix B, Behr J-P (1995) A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc Natl Acad Sci U S A 92:7297–7301
Boyce FM, Bucher NLR (1996) Baculovirus-mediated gene transfer into mammalian cells. Proc Natl Acad Sci U S A 93:2348–2352
Breuzard G, Tertil M, Gonçalves C, Cheradame H, Géguan P, Pichon C, Midoux P (2008) Nuclear delivery of NFκB-assisted DNA/polymer complexes: plasmid DNA quantitation by confocal laser scanning microscopy and evidence of nuclear polyplexes by FRET imaging. Nucleic Acids Res 36:e71
Broll S, Oumard A, Hahn K, Schambach A, Bode J (2010) Minicircle performance depending on S/MAR-nuclear matrix interactions. J Mol Biol 395:950–965
Brown AJ, Sweeney B, Mainwaring DO, James DC (2014) Synthetic promoters for CHO cell engineering. Biotechnol Bioeng 111:1638–1647
Carbonell LF, Miller LK (1987) Baculovirus interaction with nontarget organisms: a virus-borne reporter gene is not expressed in two mammalian cell lines. Appl Environ Microbiol 53:1412–1417
Carpentier E, Paris S, Kamen AA, Durocher Y (2007) Limiting factors governing protein expression following polyethylenimine-mediated gene transfer in HEK293-EBNA1 cells. J Biotechnol 128:268–280
Carter J, Zhang J, Dang T-L, Hasegawa H, Cheng JD, Gianan I, O’Neill JW, Wolfson M, Siu S, Qu S, Meininger D, Kim H, Delaney D, Mehlin C (2010) Fusion partners can increase the expression of recombinant interleukins via transient transfection in 2936E cells. Protein Sci 19:357–362
Cervera L, Gutiérrez-Granados S, Martínez M, Blanco J, Gòdia F, Segura MM (2013) Generation of HIV-1 Gag VLPs by transient transfection of HEK 293 suspension cell cultures using an optimized animal-derived component free medium. J Biotechnol 166:152–165
Chang VT, Crispin M, Aricescu AR, Harvey DJ, Nettleship JE, Fennelly JA, Yu C, Boles KS, Evans EJ, Stuart DI, Dwek RA, Jones EY, Owens RJ, Davis SJ (2007) Glycoprotein structural genomics: solving the glycosylation problem. Structure 15:267–273
Cheng L, Sun X, Yi X, Zhang Y (2011) Large-scale plasmid preparation for transient gene expression. Biotechnol Lett 33:1559–1564
Chiou HC, Vasu S, Liu CY, Cisneros I, Jones MB, Zmuda JF (2014) Scalable transient protein expression. Methods Mol Biol 1104:35–55
Cho M-S, Yee H, Brown C, Jeang K-T, Chan S (2001) An oriP expression vector containing the HIV-1 Tat/TAR transactivation axis produces high levels of protein expression in mammalian cells. Cytotechnology 37:23–30
Cho MS, Yee H, Chan S (2002) Establishment of a human somatic hybrid cell line for recombinant protein production. J Biomed Sci 9:631–638
Cho M-S, Yee H, Brown C, Mei B, Mirenda C, Chan S (2003) Versatile expression system for rapid and stable production of recombinant proteins. Biotechnol Prog 19:229–232
Chowdhury EH, Sasagawa T, Nagaoka M, Kundu AK, Akaike T (2003) Transfecting mammalian cells by DNA/calcium phosphate precipitates: effect of temperature and pH on precipitation. Anal Biochem 314:316–318
Chun B-H, Park S-Y, Chung N, Bang W-G (2003) Enhanced production of recombinant B-domain deleted factor VIII from Chinese hamster ovary cells by propionic and butyric acids. Biotechnol Lett 25:315–319
Codamo J, Munro TP, Hughes BS, Song M, Gray PP (2011) Enhanced CHO cell-based transient gene expression with the Epi-CHO expression system. Mol Biotechnol 48:109–115
Condon RGG, Schaefer EJ, Santoro M, Longley R Jr, Tsao Y-S, Zurawski SM, Liu Z (2003) Development of a Chinese hamster ovary cell line for recombinant adenovirus-mediated gene expression. Biotechnol Prog 19:137–143
Condreay JP, Witherspoon SM, Clay WC, Kost TA (1999) Transient and stable gene expression in mammalian cells transduced with a recombinant baculovirus vector. Proc Natl Acad Sci U S A 96:127–132
Corsi K, Chellat F, Yahia L’H, Fernandes JC (2003) Mesenchymal stem cells, MG63 and HEK293 transfection using chitosan-DNA nanoparticles. Biomaterials 24:1255–1264
Côté J, Garnier A, Massie B, Kamen A (1998) Serum-free production of recombinant proteins and adenoviral vectors by 293SF-3F6 cells. Biotechnol Bioeng 59:567–575
Dauty E, Verkman AS (2005) Actin cytoskeleton as the principal determinant of size-dependent DNA mobility in cytoplasm. A new barrier for non-viral gene delivery. J Biol Chem 280:7823–7828
Davies A, Greene A, Lüllau E, Abbott WM (2005) Optimisation and evaluation of a high-throughput mammalian protein expression system. Protein Expr Purif 42:111–121
Dean DA, Strong DD, Zimmer WE (2005) Nuclear entry of nonviral vectors. Gene Ther 12:881–890
Delegrange F, Matasci M, Baldi L, Wurm FM (2012) Galectin-3 overexpression enhances survival and recombinant protein expression in mammalian cells. In: Jenkins N, Barron N, Alves PM (eds) ESACT proceedings 5. Springer, Dordrecht, pp 31–35
Derouazi M, Girard P, van Tilborgh F, Iglesias K, Muller N, Bertschinger M, Wurm FM (2004) Serum-free large-scale transient transfection of CHO cells. Biotechnol Bioeng 87:537–545
Durocher Y (2006) Expression vectors containing a truncated Epstein Barr Nuclear Antigen 1 lacking the Gly-Gly-Ala domain for enhanced transient gene expression. WO2006096989
Durocher Y, Loignon M (2009) Process, vectors and engineered cell lines for enhanced large-scale transfection. WO2009137911
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
Eberhardy SR, Radzniak L, Liu Z (2009) Iron (III) citrate inhibits polyethylenimine-mediated transient transfection of Chinese hamster ovary cells in serum-free medium. Cytotechnology 60:1–9
Fan S, Maguire CA, Ramirez SH, Bradel-Tretheway B, Sapinoro R, Sui Z, Chakraborty-Sett S, Dewhurst S (2005) Valproic acid enhances gene expression from viral gene transfer vectors. J Virol Methods 125:23–33
Fath S, Bauer AP, Liss M, Spriestersbach A, Maertens B, Hahn P, Ludwig C, Schäfer F, Graf M, Wagner R (2011) Multiparameter RNA and codon optimization: a standardized tool to assess and enhance autologous mammalian gene expression. PLoS One 6:e17596
Felgner PL, Gadek TR, Holm M, Roman R, Chan HW, Wenz M, Northrop JP, Ringold GM, Danielsen M (1987) Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc Natl Acad Sci U S A 84:7413–7417
Fernández Núñez EG, Calil Jorge SA, Mancini Astray R, Gonçalves de Rezende A, Labate Vale da Costa B, Ventini Monteiro DC, Augusto Pereira C, Tonso A (2013) Semliki Forest Virus as a vector: pros and cons for its use in biopharmaceuticals production. Braz Arch Biol Technol 56:859–866
Fischer S, Charara N, Gerber A, Wölfel J, Schiedner G, Voedisch B, Geisse S (2012) Transient recombinant protein expression in a human amniocyte cell line: the CAP-T® cell system. Biotechnol Bioeng 109:2250–2261
Fliedl L, Kaisermayer C (2011) Transient gene expression in HEK293 and vero cells immobilised on microcarriers. J Biotechnol 153:15–21
Gabrielson NP, Pack DW (2009) Efficient polyethylenimine-mediated gene delivery proceeds via a caveolar pathway in HeLa cells. J Control Release 136:54–61
Galbraith DJ, Tait AS, Racher AJ, Birch JR, James DC (2006) Control of culture environment for improved polyethylenimine-mediated transient production of recombinant monoclonal antibodies by CHO cells. Biotechnol Prog 22:753–762
Gehrke R, Heinz FX, Davis NL, Mandl CW (2005) Heterologous gene expression by infectious and replicon vectors derived from tick-borne encephalitis virus and direct comparison of this flavivirus system with an alphavirus replicon. J Gen Virol 86:1045–1053
Geisse S (2009) Reflections on more than 10 years of TGE approaches. Protein Expr Purif 64:99–107
Geisse S, Henke M (2005) Large-scale transient transfection of mammalian cells: a newly emerging attractive option for recombinant protein production. J Struct Funct Genomics 6:165–170
Geng Z-H, Nudson W, Davis L, Luo S, Etchberger K (2007) Optimizing medium for transient transfection. In: Smith R (ed) Cell technology for cell products. Springer, Dordrecht, pp 53–55
Gill DR, Pringle IA, Hyde SC (2009) Progress and prospects: the design and production of plasmid vectors. Gene Ther 16:165–171
Girard P, Porte L, Berta T, Jordan M, Wurm FM (2001) Calcium phosphate transfection optimization for serum-free suspension culture. Cytotechnology 35:175–180
Girard P, Derouazi M, Baumgartner G, Bourgeois M, Jordan M, Jacko B, Wurm FM (2002) 100-Liter transient transfection. Cytotechnology 38:15–21
Godbey WT, Wu KK, Mikos AG (1999a) Size matters: molecular weight affects the efficiency of poly(ethylenimine) as a gene delivery vehicle. J Biomed Mater Res 45:268–275
Godbey WT, Wu KK, Mikos AG (1999b) Tracking the intracellular path of poly(ethylenimine)/DNA complexes for gene delivery. Proc Natl Acad Sci U S A 96:5177–5181
Golzio M, Teissié J, Rols M-P (2002) Cell synchronization effect on mammalian cell permeabilization and gene delivery by electric field. Biochim Biophys Acta 1563:23–28
Gonzalez H, Hwang SJ, Davis ME (1999) New class of polymers for the delivery of macromolecular therapeutics. Bioconjug Chem 10:1068–1074
Gorman CM, Howard BH (1983) Expression of recombinant plasmids in mammalian cells is enhanced by sodium butyrate. Nucleic Acids Res 11:7631–7648
Goula D, Remy JS, Erbacher P, Wasowicz M, Levi G, Abdallah B, Demeneix BA (1998a) Size, diffusibility and transfection performance of linear PEI/DNA complexes in the mouse central nervous system. Gene Ther 5:712–717
Goula D, Benoist C, Mantero S, Merlo G, Levi G, Demeneix BA (1998b) Polyethylenimine-based intravenous delivery of transgenes into mouse lung. Gene Ther 5:1291–1295
Graham FL, van der Eb AJ (1973) A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52:456–467
Grosjean F, Batard P, Jordan M, Wurm FM (2002) S-phase synchronized CHO cells show elevated transfection efficiency and expression using CaPi. Cytotechnology 38:57–62
Grosjean F, Bertschinger M, Hacker DL, Wurm FM (2006) Multiple glycerol shocks increase the calcium phosphate transfection of non-synchronized CHO cells. Biotechnol Lett 28:1827–1833
Grosse S, Thévenot G, Monsigny M, Fajac I (2006) Which mechanism for nuclear import of plasmid DNA complexed with polyethylenimine derivatives? J Gene Med 8:845–851
Haldankar R, Li D, Saremi Z, Baikalov C, Deshpande R (2006) Serum-free suspension large-scale transient transfection of CHO cells in WAVE bioreactors. Mol Biotechnol 34:191–199
Havenga MJE, Holterman L, Melis I, Smits S, Kaspers J, Heemskerk E, van der Vlugt R, Koldijk M, Schouten GM, Hateboer G, Brouwer K, Vogels R, Goudsmit J (2008) Serum-free transient protein production system based on adenoviral vector and PER.C6 technology: high yield and preserved bioactivity. Biotechnol Bioeng 100:273–283
Hebbar PB, Archer TK (2003) Chromatin remodeling by nuclear receptors. Chromosoma 111:495–504
Hu Q, Wang J, Shen J, Liu M, Jin X, Tang G, Chu PK (2012) Intracellular pathways and nuclear localization signal peptide-mediated gene transfection by cationic polymeric nanovectors. Biomaterials 33:1135–1145
Hu Y, Zhou D, Li C, Zhou H, Chen J, Zhang Z, Guo T (2013) Gene delivery of PEI incorporating with functional block polymer via non-covalent assembly strategy. Acta Biomater 9:5003–5012
Hunter AC (2006) Molecular hurdles in polyfectin design and mechanistic background to polycation induced cytotoxicity. Adv Drug Deliv Rev 58:1523–1531
Hust M, Meyer T, Voedisch B, Rülker T, Thie H, El-Ghezal A, Kirsch MI, Schütte M, Helmsing S, Meier D, Schirrmann T, Dübel S (2011) A human scFv antibody generation pipeline for proteome research. J Biotechnol 152:159–170
Jäger V, Büssow K, Wagner A, Weber S, Hust M, Frenzel A, Schirrmann T (2013) High level transient production of recombinant antibodies and antibody fusion proteins in HEK293 cells. BMC Biotechnol 13:52
Johansson T, Norris T, Peilot-Sjögren H (2013) Yellow fluorescent protein-based assay to measure GABAA channel activation and allosteric modulation in CHO-K1 cells. PLoS One 8(3):e59429
Jordan M, Köhne C, Wurm FM (1998) Calcium-phosphate mediated DNA transfer into HEK-293 cells in suspension: control of physicochemical parameters allows transfection in stirred media. Cytotechnology 26:39–47
Kadlecova Z, Nallet S, Hacker DL, Baldi L, Klok H-A, Wurm FM (2012) Poly(ethyleneimine)-mediated large-scale transient gene expression: influence of molecular weight, polydispersity and N-propionyl groups. Macromol Biosci 12:628–636
Kaufmann H, Mazur X, Fussenegger M, Bailey JE (1999) Influence of low temperature on productivity, proteome and protein phosphorylation of CHO cells. Biotechnol Bioeng 63:573–582
Kichler A, Leborgne C, Coeytaux E, Danos O (2001) Polyethylene-mediated gene delivery: a mechanistic study. J Gene Med 3:135–144
Kichler A, Leborgne C, Danos O (2005) Dilution of reporter gene with stuffer DNA does not alter the transfection efficiency of polyethylenimines. J Gene Med 7:1459–1467
Kim K-S, Kim MS, Moon JH, Jeong MS, Kim J, Lee GM, Myung P-K, Hong HJ (2009) Enhancement of recombinant antibody production in HEK 293E cells by WPRE. Biotechnol Bioproc Eng 14:633–638
Kiseljak D, Rajendra Y, Manoli SS, Baldi L, Hacker DL, Wurm FM (2011) The use of filler DNA for improved transfection and reduced DNA needs in transient gene expression with CHO and HEK cells. BMC Proc 5(Suppl 8):P33
Kiseljak D, Rajendra Y, Backliwal G, Hacker DL, Baldi L, Wurm FM (2012) Recombinant antibody yield over 2 g/L by transient transfection of HEK 293 EBNA cells in a fed-batch process. In: Jenkins N, Barron N, Alves PM (eds) ESACT proceedings 5. Springer, Dordrecht, pp 497–500
Kunaparaju R, Liao M, Sunstrom N-A (2005) Epi-CHO, an episomal expression system for recombinant protein production in CHO cells. Biotechnol Bioeng 91:670–677
Labat-Moleur F, Steffan A-M, Brisson C, Perron H, Feugeas O, Furstenberger P, Oberling F, Brambilla E, Behr J-P (1996) An electron microscopy study into the mechanism of gene transfer with lipopolyamines. Gene Ther 3:1010–1017
Längle-Rouault F, Patzel V, Benavente A, Taillez M, Silvestre N, Bompard A, Sczakiel G, Jacobs E, Rittner K (1998) Up to 100-fold increase of apparent gene expression in the presence of Epstein-Barr virus oriP sequences and EBNA1: implications of the nuclear import of plasmids. J Virol 72:6181–6185
Lechardeur D, Sohn K-J, Haardt M, Joshi PB, Monck M, Graham RW, Beatty B, Squire J, O’Brodovich H, Lukacs GL (1999) Metabolic instability of plasmid DNA in the cytosol: a potential barrier to gene transfer. Gene Ther 6:482–497
Lee JE, Fusco ML, Ollmann Saphire E (2009) An efficient platform for screening expression and crystallization of glycoproteins produced in human cells. Nat Protoc 4:592–604
Legendre JY, Trzeciak A, Bohrmann B, Deuschle U, Kitas E, Supersaxo A (1997) Dioleoylmelittin as a novel serum-insensitive reagent for efficient transfection of mammalian cells. Bioconjug Chem 8:57–63
Li J, Menzel C, Meier D, Zhang C, Dübel S, Jostock T (2007) A comparative study of different vector designs for the mammalian expression of recombinant IgG antibodies. J Immunol Methods 318:113–124
Lindell J, Girard P, Müller N, Jordan M, Wurm FM (2004) Calfection: a novel gene transfer method for suspension cells. Biophys Biochim Acta 1676:155–161
Lindner SE, Sugden B (2007) The plasmid replicon of Epstein-Barr virus: mechanistic insights into efficient, licensed, extrachromosomal replication in human cells. Plasmid 58:1–12
Liu C-H, Chu I-M, Hwang S-M (2001) Pentanoic acid, a novel protein synthesis stimulant for Chinese Hamster Ovary (CHO) cells. J Biosci Bioeng 91:71–75
Liu C, Dalby B, Chen W, Kilzer JM, Chiou HC (2008) Transient transfection factors for high-level recombinant protein production in suspension cultured mammalian cells. Mol Biotechnol 39:141–153
Liu J, Jiang Z, Zhou J, Zhang S, Saltzman WM (2011) Enzyme-synthesized poly(amine-co-esters) as non-viral vectors for gene delivery. J Biomed Mater Res A 96:456–465
Loignon M, Perret S, Kelly J, Boulais D, Cass B, Bisson L, Afkhamizarreh F, Durocher Y (2008) Stable high volumetric production of glycosylated human recombinant IFNalpha2b in HEK293 cells. BMC Biotechnol 8:65
Lukacs GL, Haggie P, Seksek O, Lechardeur D, Freedman N, Verkman AS (2000) Size-dependent DNA mobility in cytoplasm and nucleus. J Biol Chem 275:1625–1629
Luke J, Carnes AE, Hodgson CP, Williams JA (2009) Improved antibiotic-free DNA vaccine vectors utilizing a novel RNA based plasmid selection system. Vaccine 27:6454–6459
Lundstrom K (2010) Expression of mammalian membrane proteins in mammalian cells using Semliki Forest Virus vectors. Methods Mol Biol 601:149–163
Mairhofer J, Grabherr R (2008) Rational vector design for efficient non-viral gene delivery: challenges facing the use of plasmid DNA. Mol Biotechnol 39:97–104
Mairhofer J, Pfaffenzeller I, Merz D, Grabherr R (2008) A novel antibiotic free plasmid selection system: advances in safe and efficient DNA therapy. Biotechnol J 3:83–89
Majors BS, Betenbaugh MJ, Pederson NE, Chiang GC (2008) Enhancement of transient gene expression and culture viability using Chinese Hamster Ovary cells overexpressing Bcl-xL. Biotechnol Bioeng 101:567–578
Mariati YKN, Chao S-H, Yap MGS, Yang Y (2010) Evaluating regulatory elements of human cytomegalovirus major immediate early gene for enhancing transgene expression levels in CHO K1 and HEK293 cells. J Biotechnol 147:160–163
Marie C, Quiviger M, Foster H, Dickson G, Scherman D (2013) Plasmid-based medicinal products – focus on pFAR: a miniplasmid free of antibiotic resistance markers. In: Schleef M (ed) Minicircle and miniplasmid DNA vectors. Wiley-Blackwell, Weinheim, pp 37–58
Meissner P, Pick H, Kulangara A, Chatellard P, Friedrich K, Wurm FM (2001) Transient gene expression: recombinant protein production with suspension-adapted HEK293-EBNA cells. Biotechnol Bioeng 75:197–203
Melkonyan H, Sorg C, Klempt M (1996) Electroporation efficiency in mammalian cells is increased by dimethyl sulfoxide (DMSO). Nucleic Acids Res 24:4356–4357
Nehlsen K, Broll S, Kandimalla R, Heinz N, Heine M, Binius S, Schambach A, Bode J (2013) Replicating minicircles: overcoming the limitations of transient and stable expression systems. In: Schleef M (ed) Minicircle and miniplasmid DNA vectors. Wiley-Blackwell, Weinheim, pp 115–164
Nel AE, Mädler L, Velegol D, Xia T, Hoek EMV, Somasundaran P, Klaessig F, Castranova V, Thompson M (2009) Understanding the biophysicochemical interactions at the nano-bio interface. Nat Mater 8:543–557
Nivitchanyong T, Tsai YC, Betenbaugh MJ, Oyler GA (2009) An improved in vitro and in vivo Sindbis virus expression system through host and virus engineering. Virus Res 141:1–12
Ogawa R, Kagiya G, Kodaki T, Fukuda S, Yamamoto K (2007) Construction of strong mammalian promoters by random cis-acting element elongation. Biotechniques 42:628–633
Oh Y-K, Suh D, Kim JM, Choi H-G, Ko JJ (2002) Polyethylenimine-mediated cellular uptake, nucleus trafficking and expression of cytokine plasmid DNA. Gene Ther 9:1627–1632
Oliveira PH, Mairhofer J (2013) Marker-free plasmids for biotechnological applications – implications and perspectives. Trends Biotechnol 31:539–547
Oliveira PH, Prather KJ, Prazeres DM, Monteiro GA (2009) Structural instability of plasmid biopharmaceuticals: challenges and implications. Trends Biotechnol 27:503–511
Parham JH, Iannone MA, Overton LK, Hutchins JT (1998) Optimization of transient gene expression in mammalian cells and potential for scale-up using flow electroporation. Cytotechnology 28:147–155
Parham JH, Kost T, Hutchins JT (2001) Effects of pCIneo and pCEP4 expression vectors on transient and stable protein production in human and simian cell lines. Cytotechnology 35:181–187
Paris S, Burlacu A, Durocher Y (2007) Opposing roles of syndecan-1 and syndecan-2 in polyethylenimine-mediated gene delivery. J Biol Chem 283:7697–7704
Payne CK, Jones SA, Chen C, Zhuang X (2007) Internalization and trafficking of cell surface proteoglycans and proteoglycan-binding ligands. Traffic 8:389–401
Pear WS, Nolan GP, Scott ML, Baltimore D (1993) Production of high-titer helper-free retroviruses by transient transfection. Proc Natl Acad Sci U S A 90:8392–8396
Pereira J, Rajendra Y, Baldi L, Hacker DL, Wurm FM (2011) Transient gene expression with CHO cells in conditioned medium: a study using TubeSpin® bioreactors. BMC Proc 5(Suppl 8):P38
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
Pham PL, Perret S, Cass B, Carpentier E, St-Laurent G, Bisson L, Kamen A, Durocher Y (2005) Transient gene expression in HEK293 cells: peptone addition posttransfection improves recombinant protein synthesis. Biotechnol Bioeng 90:332–344
Pham PL, Kamen A, Durocher Y (2006) Large-scale transfection of mammalian cells for the fast production of recombinant protein. Mol Biotechnol 34:225–237
Phez E, Faurie C, Golzio M, Teissié J, Rols M-P (2005) New insights in the visualization of membrane permeabilization and DNA/membrane interaction of cells submitted to electric pulses. Biochim Biophys Acta 1724:248–254
Pick HM, Meissner P, Preuss AK, Tromba P, Vogel H, Wurm FM (2002) Balancing GFP reporter plasmid quantity in large-scale transient transfections for recombinant anti-human Rhesus-D IgG1 synthesis. Biotechnol Bioeng 79:595–601
Polo JM, Belli BA, Driver DA, Frolov I, Sherrill S, Hariharan MJ, Townsend K, Perri S, Mento SJ, Jolly DJ, Chang SMW, Schlesinger S, Dubensky TW Jr (1999) Stable alphavirus packaging cell lines for Sindbis virus- and Semliki Forest virus-derived vectors. Proc Natl Acad Sci U S A 96:4598–4603
Pradeau-Aubreton K, Ruff M, Garnier J-M, Schultz P, Drillien R (2010) Vectors for recombinational cloning and gene expression in mammalian cells using modified vaccinia virus Ankara. Anal Biochem 404:103–105
Rajendra Y, Kiseljak D, Baldi L, Hacker DL, Wurm FM (2011) A simple high-yielding process for transient gene expression in CHO cells. J Biotechnol 153:22–26
Rajendra Y, Kiseljak D, Manoli S, Baldi L, Hacker DL, Wurm FM (2012) Role of non-specific DNA in reducing coding DNA requirement for transient gene expression with CHO and HEK-293E cells. Biotechnol Bioeng 109:2271–2278
Raymond C, Tom R, Perret S, Moussouami P, L’Abbé D, St-Laurent G, Durocher Y (2011) A simplified polyethylenimine-mediated transfection process for large-scale and high-throughput applications. Methods 55:44–51
Reed SE, Staley EM, Mayginnes JP, Pintel DJ, Tullis GE (2006) Transfection of mammalian cells using linear polyethylenimine is a simple and effective means of producing recombinant adeno-associated virus vectors. J Virol Methods 138:85–98
Reeves PJ, Callewaert N, Contreras R, Khorana HG (2002) Structure and function in rhodopsin: high-level expression of rhodopsin with restricted and homogeneous N-glycosylation by a tetracyclineinducible N-acetylglucosaminyltransferase I-negative HEK293S stable mammalian cell line. Proc Natl Acad Sci U S A 99:13419–13424
Rols M-P, Teissié J (1992) Experimental evidence for the involvement of the cytoskeleton in mammalian cell electropermeabilization. Biochim Biophys Acta 1111:45–50
Rosazza C, Buntz A, Rieß T, Wöll D, Zumbusch A, Rols M-P (2013) Intracellular tracking of single-plasmid DNA particles after delivery by electroporation. Mol Ther 21:2217–2226
Rosser MP, Xia W, Hartsell S, McCaman M, Zhu Y, Wang S, Harvey S, Bringmann P, Cobb RR (2005) Transient transfection of CHO-K1-S using serum-free medium in suspension: a rapid mammalian protein expression system. Protein Expr Purif 40:237–243
Rozkov A, Larsson B, Gillström S, Björnestedt R, Schmidt SR (2008) Large-scale production of endotoxin-free plasmids for transient gene expression in mammalian cell culture. Biotechnol Bioeng 99:557–566
Schambach A, Wodrich H, Hildinger M, Bohne J, Krausslich H-G, Baum C (2000) Context dependence of different modules for posttranscriptional enhancement of gene expression from retroviral vectors. Mol Ther 2:435–445
Schirrmann T, Büssow K (2010) Transient production of scFv-Fc fusion proteins in mammalian cells. In: Kontermann R, Dübel S (eds) Antibody engineering, vol 2, 2nd edn. Springer, Heidelberg, pp 387–400
Schlabach MR, Hu JK, Li M, Elledge SJ (2010) Synthetic design of strong promoters. Proc Natl Acad Sci U S A 107:2538–2543
Schlaeger E-J, Christensen K (1999) Transient gene expression in mammalian cells grown in serum-free suspension culture. Cytotechnology 30:71–83
Schlaeger E-J, Lundstrom K (1998) Effect of temperature on recombinant protein expression in Semliki Forest virus infected mammalian cell lines growing in serum-free suspension cultures. Cytotechnology 28:205–211
Schlaeger E-J, Kitas EA, Dorn A (2003) SEAP expression in transiently transfected mammalian cells grown in serum-free suspension culture. Cytotechnology 42:47–55
Shi J, Chou B, Choi JL, Ta AL, Pun SH (2013) Investigation of polyethylenimine/DNA polyplex transfection to cultured cells using radiolabeling and subcellular fractionation methods. Mol Pharm 10:2145–2156
Silla T, Tagen I, Geimanen J, Janikson K, Abroi A, Ustav E, Ustav M, Mandel T (2007) Vectors, cell lines and their use in obtaining extended episomal maintenance replication of hybrid plasmids and expression of gene products. WO2006084754
Silla T, Tagen I, Kalling A, Tegova R, Ustav M, Mandel T, Toots U, Tover A, Abroi A, Ustav E, Geimanen J, Janikson K (2011) Viral expression plasmids for production of proteins, antibodies, enzymes, virus-like particles and for use in cell-based assays. US20110076760
Smerdou C, Liljeström P (2000) Alphavirus vectors: highly efficient systems for transient gene expression. In: Al-Rubeai M (ed) Cell engineering, vol 2, Transient expression. Kluwer, Dordrecht, pp 182–210
Stettler M, Zhang X, Hacker DL, De Jesus M, Wurm FM (2007) Novel orbital shake bioreactors for transient production of CHO derived IgGs. Biotechnol Prog 23:1340–1346
Stoops J, Byrd S, Hasegawa H (2012) Russell body inducing threshold depends on the variable domain sequences of individual human IgG clones and the cellular protein homeostasis. Biochim Biophys Acta 1823:1643–1657
Sun X, Goh PE, Wong KTK, Mori T, Yap MGS (2006) Enhancement of transient gene expression by fed-batch culture of HEK 293 EBNA1 cells in suspension. Biotechnol Lett 28:843–848
Sun X, Hia HC, Goh PE, Yap MGS (2008) High-density transient gene expression in suspension-adapted 293 EBNA1 cells. Biotechnol Bioeng 99:108–116
Swiech K, Kamen A, Ansorge S, Durocher Y, Picanço-Castro V, Russo-Carbolante EMS, Neto MSA, Covas DT (2011) Transient transfection of serum-free suspension HEK 293 cell culture for efficient production of human rFVIII. BMC Biotechnol 11:114
Tait AS, Brown CJ, Galbraith DJ, Hines MJ, Hoare M, Birch JR, James DC (2004) Transient production of recombinant proteins by Chinese Hamster Ovary cells using Polyethyleneimine/DNA complexes in combination with microtubule disrupting anti-mitotic agents. Biotechnol Bioeng 88:707–721
Tuvesson O, Uhe C, Rozkov A, Lüllau E (2008) Development of a generic transient transfection process at 100 L scale. Cytotechnology 56:123–136
ur Rehman Z, Sjollema KA, Kuipers J, Hoekstra D, Zuhorn IS (2012) Nonviral gene delivery vectors use syndecan-dependent transport mechanisms in filopodia to reach the cell surface. ACS Nano 6:7521–7532
ur Rehman Z, Hoekstra D, Zuhorn IS (2013) Mechanism of polyplex- and lipoplex-mediated delivery of nucleic acids: real-time visualization of transient membrane destabilization without endosomal lysis. ACS Nano 7:3767–3777
van der Aa MAEM, Mastrobattista E, Oosting RS, Hennink WE, Koning GA, Crommelin DJA (2006) The nuclear pore complex: the gateway to successful nonviral gene delivery. Pharm Res 23:447–459
van der Aa MAEM, Huth US, Häfele SY, Schubert R, Oosting RS, Mastrobattista E, Hennink WE, Peschka-Süss R, Koning GA, Crommelin DJA (2007) Cellular uptake of cationic polymer-DNA complexes via caveolae plays a pivotal role in gene transfection in COS-7 cells. Pharm Res 24:1590–1598
Vasu SK, Chiou HC, Rogers J, Cisneros M, Li J, Liu CY, Jones M (2013) High yield transient expression in mammalian cells using unique pairing of high density growth and transfection medium and expression enhancers. WO2013166339
von Gersdorff K, Sanders NN, Vandenbroucke R, De Smedt SC, Wagner E, Ogris M (2006) The internalization route resulting in successful gene expression depends on both cell line and polyethylenimine polyplex type. Mol Ther 14:745–753
Wang Y, Zheng M, Meng F, Zhang J, Peng R, Zhong Z (2011) Branched polyethylenimine derivatives with reductively cleavable periphery for safe and efficient in vitro gene transfer. Biomacromolecules 12:1032–1040
Weidemann R, Ludwig A, Kretzmer G (1994) Low temperature cultivation – a step towards process optimization. Cytotechnology 15:111–116
Williams SG, Cranenburgh RM, Weiss AM, Wrighton CJ, Sherratt DJ, Hanak JA (1998) Repressor titration: a novel system for selection and stable maintenance of recombinant plasmids. Nucleic Acids Res 26:2120–2124
Wiseman JW, Goddard CA, McLelland D, Colledge WH (2003) A comparison of linear and branched polyethylenimine (PEI) with DCChol/DOPE liposomes for gene delivery to epithelial cells in vitro and in vivo. Gene Ther 10:1654–1662
Wölfel J, Essers R, Bialek C, Hertel S, Scholz-Neumann N, Schiedner G (2011) CAP-T cell expression system: a novel rapid and versatile human cell expression system for fast and high yield transient protein expression. BMC Proc 5(Suppl 8):P133
Wong T-K, Neumann E (1982) Electric field mediated gene transfer. Biochem Biophys Res Commun 107:584–587
Wulhfard S, Tissot S, Bouchet S, Cevey J, De Jesus M, Hacker DL, Wurm FM (2008) Mild hypothermia improves transient gene expression yields several fold in Chinese Hamster Ovary cells. Biotechnol Prog 24:458–465
Xia W, Bringmann P, McClary J, Jones PP, Manzana W, Zhu Y, Wang S, Liu Y, Harvey S, Madlansacay MR, McLean K, Rosser MP, MacRobbie J, Olsen CL, Cobb RR (2006) High levels of protein expression using different mammalian CMV promoters in several cell lines. Protein Expr Purif 45:115–124
Yamano S, Dai J, Moursi AM (2010) Comparison of transfection efficiency of nonviral gene transfer reagents. Mol Biotechnol 46:287–300
Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103–119
Yates JL, Warren N, Sugden B (1985) Stable replication of plasmids derived from Epstein-Barr virus in various mammalian cells. Nature 313:812–815
Ye J, Kober V, Tellers M, Naji Z, Salmon P, Markusen JF (2009) High-level protein expression in scalable CHO transient transfection. Biotechnol Bioeng 103:542–551
Yin W, Xiang P, Li Q (2005) Investigations of the effect of DNA size in transient transfection assay using dual luciferase system. Anal Biochem 346:289–294
Yue Y, Jin F, Deng R, Cai J, Dai Z, Lin MCM, Kung H-F, Mattebjerg MA, Andresen TL, Wu C (2011) Revisit complexation between DNA and polyethylenimine – effect of length of free polycationic chains on gene transfection. J Control Release 152:143–151
Yun S-I, Kim S-Y, Rice CM, Lee Y-M (2003) Development and amplification of a reverse genetics system for Japanese Encephalitis Virus. J Virol 77:6450–6465
Zabner J, Fasbender AJ, Moninger T, Poellinger KA, Welsh MJ (1995) Cellular and molecular barriers to gene transfer by a cationic lipid. J Biol Chem 270:18997–19007
Zhabinskaya D, Benham CJ (2011) Theoretical analysis of the stress induced B-Z transition in superhelical DNA. PLoS Comput Biol 7:e1001051
Zhang J, Liu X, Bell A, To R, Baral TN, Azizi A, Li J, Cass B, Durocher Y (2009) Transient expression and purification of chimeric heavy chain antibodies. Protein Expr Purif 65:77–82
Zhao Q-Q, Chen J-L, Lv T-F, He C-X, Tang G-P, Liang W-Q, Tabata Y, Gao J-Q (2009) N/P ratio significantly influences the transfection efficiency and cytotoxicity of a polyethylenimine/chitosan/DNA complex. Biol Pharm Bull 32:706–710
Zhao Y, Bishop B, Clay JE, Lu W, Jones M, Daenke S, Siebold C, Stuart DI, Jones EY, Aricescu AR (2011) Automation of large scale transient protein expression in mammalian cells. J Struct Biol 175:209–215
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2015 Springer International Publishing Switzerland
About this chapter
Cite this chapter
Jäger, V., Büssow, K., Schirrmann, T. (2015). Transient Recombinant Protein Expression in Mammalian Cells. In: Al-Rubeai, M. (eds) Animal Cell Culture. Cell Engineering, vol 9. Springer, Cham. https://doi.org/10.1007/978-3-319-10320-4_2
Download citation
DOI: https://doi.org/10.1007/978-3-319-10320-4_2
Published:
Publisher Name: Springer, Cham
Print ISBN: 978-3-319-10319-8
Online ISBN: 978-3-319-10320-4
eBook Packages: Biomedical and Life SciencesBiomedical and Life Sciences (R0)