A Novel Terminator Primer and Enhancer Reagents for Direct Expression of PCR-Amplified Genes in Mammalian Cells
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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.
KeywordsTransfection enhancer Terminator primer Linear DNA Polyethylene glycol Transfer RNA (tRNA) PCR Oligonucleotide primer Expression construct
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.
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