title: A Novel Terminator Primer and Enhancer Reagents for Direct Expression of PCR-Amplified Genes in Mammalian Cells.
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.
authors.affiliation: Innovation Center, Yamaguchi University, Tokiwadai, Ube, 755-8611, Japan, nakamura@crc.yamaguchi-u.ac.jp.
authors.forename: Mikiko
authors.initials: M
authors.lastname: Nakamura
authors.forename: Ayako
authors.initials: A
authors.lastname: Suzuki
authors.forename: Junko
authors.initials: J
authors.lastname: Akada
authors.forename: Tohru
authors.initials: T
authors.lastname: Yarimizu
authors.forename: Ryo
authors.initials: R
authors.lastname: Iwakiri
authors.forename: Hisashi
authors.initials: H
authors.lastname: Hoshida
authors.forename: Rinji
authors.initials: R
authors.lastname: Akada
mesh_terms: D000818:Animals; D017931:DNA Primers / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D010957:Plasmids / Q000737:chemistry* / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D013728:Terminator Regions, Genetic; D014162:Transfection
chemical_list: D017931:DNA Primers; D011994:Recombinant Proteins