Abstract
Escherichia coli plasmids are commonly used for gene expression experiments in mammalian cells, while PCR-amplified DNAs are rarely used even though PCR is a much faster and easier method to construct recombinant DNAs. One difficulty may be the limited amount of DNA produced by PCR. For direct utilization of PCR-amplified DNA in transfection experiments, efficient transfection with a smaller amount of DNA should be attained. For this purpose, we investigated two enhancer reagents, polyethylene glycol and tRNA, for a chemical transfection method. The addition of the enhancers to a commercial transfection reagent individually and synergistically exhibited higher transfection efficiency applicable for several mammalian cell culture lines in a 96-well plate. By taking advantage of a simple transfection procedure using PCR-amplified DNA, SV40 and rabbit β-globin terminator lengths were minimized. The terminator length is short enough to design in oligonucleotides; thus, terminator primers can be used for the construction and analysis of numerous mutations, deletions, insertions, and tag-fusions at the 3′-terminus of any gene. The PCR-mediated gene manipulation with the terminator primers will transform gene expression by allowing for extremely simple and high-throughput experiments with small-scale, multi-well, and mammalian cell cultures.
Similar content being viewed by others
References
Witzgall, R., O’Leary, E., & Bonventre, J. V. (1994). A mammalian expression vector for the expression of GAL4 fusion proteins with an epitope tag and histidine tail. Analytical Biochemistry, 223, 291–298.
Lejard, V., Rebours, E., Meersseman, C., & Rocha, D. (2014). Construction and validation of a novel dual reporter vector for studying mammalian bidirectional promoters. Plasmid, 74, 1–8.
Elmileik, H., Kumagai, T., Berengena, M., Ueda, K., & Sugiyama, M. (2001). Use of bleomycin- and heat shock-induced calreticulin promoter for construction of a mammalian expression vector. Journal of Biochemistry, 129, 671–674.
Roure, A., Rothbacher, U., Robin, F., Kalmar, E., Ferone, G., Lamy, C., et al. (2007). A multicassette gateway vector set for high throughput and comparative analyses in ciona and vertebrate embryos. PLoS One, 2, e916.
Tan, R., Li, C., Jiang, S., & Ma, L. (2006). A novel and simple method for construction of recombinant adenoviruses. Nucleic Acids Research, 34, e89.
Matsumoto, A., & Itoh, T. Q. (2011). Self-assembly cloning: a rapid construction method for recombinant molecules from multiple fragments. BioTechniques, 51, 55–56.
You, L. M., Luo, J., Wang, A. P., Zhang, G. P., Weng, H. B., Guo, Y. N., et al. (2010). A hybrid promoter-containing vector for direct cloning and enhanced expression of PCR-amplified ORFs in mammalian cells. Molecular Biology Reports, 37, 2757–2765.
Sugiyama, H., Niwa, H., Makino, K., & Kakunaga, T. (1988). Strong transcriptional promoter in the 5′ upstream region of the human beta-actin gene. Gene, 65, 135–139.
Wilkinson, G. W., & Akrigg, A. (1992). Constitutive and enhanced expression from the CMV major IE promoter in a defective adenovirus vector. Nucleic Acids Research, 20, 2233–2239.
Teschendorf, C., Warrington, K. H, Jr, Siemann, D. W., & Muzyczka, N. (2002). Comparison of the EF-1 alpha and the CMV promoter for engineering stable tumor cell lines using recombinant adeno-associated virus. Anticancer Research, 22, 3325–3330.
Jianwei, D., Qianqian, Z., Songcai, L., Mingjun, Z., Xiaohui, R., Linlin, H., et al. (2012). The combination of a synthetic promoter and a CMV promoter improves foreign gene expression efficiency in myocytes. Journal of Biotechnology, 158, 91–96.
Cab-Barrera, E. L., & Barrera-Saldana, H. A. (1988). Versatile plasmid vectors for use in studies of eukaryotic gene expression. Gene, 70, 411–413.
Kim, D., Kim, J. D., Baek, K., Yoon, Y., & Yoon, J. (2003). Improved mammalian expression systems by manipulating transcriptional termination regions. Biotechnology Progress, 19, 1620–1622.
Falck-Pedersen, E., Logan, J., Shenk, T., & Darnell, J. E, Jr. (1985). Transcription termination within the E1A gene of adenovirus induced by insertion of the mouse beta-major globin terminator element. Cell, 40, 897–905.
Petitclerc, D., Attal, J., Theron, M. C., Bearzotti, M., Bolifraud, P., Kann, G., et al. (1995). The effect of various introns and transcription terminators on the efficiency of expression vectors in various cultured cell lines and in the mammary gland of transgenic mice. Journal of Biotechnology, 40, 169–178.
Fritze, C. E., & Anderson, T. R. (2000). Epitope tagging: general method for tracking recombinant proteins. Methods in Enzymology, 327, 3–16.
Bell, M. R., Engleka, M. J., Malik, A., & Strickler, J. E. (2013). To fuse or not to fuse: what is your purpose? Protein Science : A Publication of the Protein Society, 22, 1466–1477.
Zhao, X., Li, G., & Liang, S. (2013). Several affinity tags commonly used in chromatographic purification. Journal of Analytical Methods in Chemistry, 2013, 581093.
Naylor, L. H. (1999). Reporter gene technology: the future looks bright. Biochemical Pharmacology, 58, 749–757.
Welsh, D. K., & Kay, S. A. (2005). Bioluminescence imaging in living organisms. Current Opinion in Biotechnology, 16, 73–78.
Prescher, J. A., & Contag, C. H. (2010). Guided by the light: visualizing biomolecular processes in living animals with bioluminescence. Current Opinion in Chemical Biology, 14, 80–89.
Jiang, T., Xing, B., & Rao, J. (2008). Recent developments of biological reporter technology for detecting gene expression. Biotechnology and Genetic Engineering Reviews, 25, 41–75.
Qureshi, S. A. (2007). Beta-lactamase: an ideal reporter system for monitoring gene expression in live eukaryotic cells. BioTechniques, 42, 91–96.
Chalfie, M. (1995). Green fluorescent protein. Photochemistry and Photobiology, 62, 651–656.
Chen, S., Zhou, D., Swiderek, K. M., Kadohama, N., Osawa, Y., & Hall, P. F. (1993). Structure-function studies of human aromatase. The Journal of Steroid Biochemistry and Molecular Biology, 44, 347–356.
Grabenhorst, E., Schlenke, P., Pohl, S., Nimtz, M., & Conradt, H. S. (1999). Genetic engineering of recombinant glycoproteins and the glycosylation pathway in mammalian host cells. Glycoconjugate Journal, 16, 81–97.
Safinya, C. R. (2001). Structures of lipid-DNA complexes: supramolecular assembly and gene delivery. Current Opinion in Structural Biology, 11, 440–448.
Li, W., & Szoka, F. C, Jr. (2007). Lipid-based nanoparticles for nucleic acid delivery. Pharmaceutical Research, 24, 438–449.
Gopalakrishnan, B., & Wolff, J. (2009). siRNA and DNA transfer to cultured cells. Methods in Molecular Biology, 480, 31–52.
Prasher, D. C. (1995). Using GFP to see the light. Trends in Genetics, 11, 320–323.
Niwa, H., Yamamura, K., & Miyazaki, J. (1991). Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene, 108, 193–199.
Okabe, M., Ikawa, M., Kominami, K., Nakanishi, T., & Nishimune, Y. (1997). ‘Green mice’ as a source of ubiquitous green cells. FEBS Letters, 407, 313–319.
Nakamura, M., Suzuki, A., Hoshida, H., & Akada, R. (2014). Minimum GC-rich sequences for overlap extension PCR and primer annealing. Methods in Molecular Biology, 1116, 165–181.
Fischer, W., Puls, J., Buhrdorf, R., Gebert, B., Odenbreit, S., & Haas, R. (2001). Systematic mutagenesis of the Helicobacter pylori cag pathogenicity island: essential genes for CagA translocation in host cells and induction of interleukin-8. Molecular Microbiology, 42, 1337–1348.
Iakhiaeva, E., Hinck, C. S., Hinck, A. P., & Zwieb, C. (2009). Characterization of the SRP68/72 interface of human signal recognition particle by systematic site-directed mutagenesis. Protein Science, 18, 2183–2195.
Burre, J., Sharma, M., & Sudhof, T. C. (2012). Systematic mutagenesis of alpha-synuclein reveals distinct sequence requirements for physiological and pathological activities. The Journal of Neuroscience, 32, 15227–15242.
Seng, K. C., & Seng, C. K. (2008). The success of the genome-wide association approach: a brief story of a long struggle. European Journal of Human Genetics, 16, 554–564.
Hsu, C. Y., & Uludag, H. (2008). Effects of size and topology of DNA molecules on intracellular delivery with non-viral gene carriers. BMC Biotechnology, 8, 23.
Dhanoya, A., Chain, B. M., & Keshavarz-Moore, E. (2011). The impact of DNA topology on polyplex uptake and transfection efficiency in mammalian cells. Journal of Biotechnology, 155, 377–386.
Remaut, K., Sanders, N. N., Fayazpour, F., Demeester, J., & De Smedt, S. C. (2006). Influence of plasmid DNA topology on the transfection properties of DOTAP/DOPE lipoplexes. Journal of Controlled Release, 115, 335–343.
Kamiya, H., Yamazaki, J., & Harashima, H. (2002). Size and topology of exogenous DNA as determinant factors of transgene transcription in mammalian cells. Gene Therapy, 9, 1500–1507.
Weintraub, H., Cheng, P. F., & Conrad, K. (1986). Expression of transfected DNA depends on DNA topology. Cell, 46, 115–122.
Cherng, J. Y., Schuurmans-Nieuwenbroek, N. M., Jiskoot, W., Talsma, H., Zuidam, N. J., Hennink, W. E., & Crommelin, D. J. (1999). Effect of DNA topology on the transfection efficiency of poly((2-dimethylamino)ethyl methacrylate)-plasmid complexes. Journal of Controlled Release, 60, 343–353.
Shen, H., Hu, Y., & Saltzman, W. M. (2006). DNA diffusion in mucus: effect of size, topology of DNAs, and transfection reagents. Biophysical Journal, 91, 639–644.
Suzuki, H., Fukunishi, Y., Kagawa, I., Saito, R., Oda, H., Endo, T., et al. (2001). Protein–protein interaction panel using mouse full-length cDNAs. Genome Research, 11, 1758–1765.
Hoat, T. X., Bertin, N., Ninomiya, N., Fukuda, S., Usui, K., Kawai, J., et al. (2009). Development of a high-throughput method for the systematic identification of human proteins nuclear translocation potential. BMC Cell Biology, 10, 69.
Xin, W., Zhang, Y. M., Xiao, J. H., & Huang, D. W. (2003). Construction of linear functional expression elements with DNA fragments created by site-specific DNA nickase, N.Bpu10 I, and exonuclease III. Biotechnology Letters, 25, 1913–1916.
Xiao, J. H., Xin, W., Liu, Y. J., Murphy, R. W., & Huang, D. W. (2007). Generation of linear expression constructs by one-step PCR with vaccinia DNA topoisomerase I. Molecular Biotechnology, 35, 15–22.
Sykes, K. F., & Johnston, S. A. (1999). Linear expression elements: a rapid, in vivo, method to screen for gene functions. Nature Biotechnology, 17, 355–359.
Susa, T., Kato, T., & Kato, Y. (2008). Reproducible transfection in the presence of carrier DNA using FuGENE6 and Lipofectamine 2000. Molecular Biology Reports, 35, 313–319.
Pradhan, K., & Gadgil, M. (2012). Effect of addition of ‘carrier’ DNA during transient protein expression in suspension CHO culture. Cytotechnology, 64, 613–622.
Ross, P. C., & Hui, S. W. (1999). Polyethylene glycol enhances lipoplex-cell association and lipofection. Biochimica et Biophysica Acta, 1421, 273–283.
Palmer, L. R., Chen, T., Lam, A. M., Fenske, D. B., Wong, K. F., MacLachlan, I., & Cullis, P. R. (2003). Transfection properties of stabilized plasmid-lipid particles containing cationic PEG lipids. Biochimica et Biophysica Acta, 1611, 204–216.
Kawai, S., & Nishizawa, M. (1984). New procedure for DNA transfection with polycation and dimethyl sulfoxide. Molecular and Cellular Biology, 4, 1172–1174.
Schiestl, R. H., & Gietz, R. D. (1989). High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Current Genetics, 16, 339–346.
Acknowledgments
We would like to thank Yukie Misumi for technical assistance. This study was supported in part by the JSPS KAKENHI (Grant Nos. 25660080, 24658096) and the Adaptable and Seamless Technology Transfer Program through Target-Driven R&D (JST, Japan), and the YU “Pump-Priming Program” for fostering Research activities.
Conflicts of interest
Mikiko Nakamura, Ayako Suzuki, Junko Akada, Tohru Yarimizu, and Hisashi Hoshida: No competing interests exist. Rinji Akada: Yamaguchi University receives grant support from KOHJIN Life Sciences for studies in which Rinji Akada serves as the principal investigator. Ryo Iwakiri: As an employee of KOHJIN Life Sciences, Ryo Iwakiri participated in the development of the transfection enhancers but not as an applicant on a patent belonging to Yamaguchi University. These associations do not alter the authors’ adherence to Molecular Biotechnology policies on sharing data and materials.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Nakamura, M., Suzuki, A., Akada, J. et al. A Novel Terminator Primer and Enhancer Reagents for Direct Expression of PCR-Amplified Genes in Mammalian Cells. Mol Biotechnol 57, 767–780 (2015). https://doi.org/10.1007/s12033-015-9870-5
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12033-015-9870-5