title: Transfection efficiency of normal and cancer cell lines and monitoring of promoter activity by single-cell bioluminescence imaging.
abstract: The bioluminescence system (luciferase reporter assay system) is widely used to study gene expression, signal transduction and other cellular activities. Although transfection of reporter plasmid DNA to mammalian cell lines is an indispensable experimental step, the transfection efficiency of DNA varies among cell lines, and several cell lines are not suitable for this type of assay because of the low transfection efficiency. In this study, we confirm the transfection efficiency of reporter DNA to several cancer and normal cell lines after transient transfection by single-cell imaging. Luminescence images could be obtained from living single cells after transient transfection, and the calculated transfection efficiency of this method was similar to that of the conventional reporter assay using a luminometer. We attempted to measure the activity of the Bip promoter under endoplasmic reticulum stress conditions using both high and low transfection efficiency cells for plasmid DNA at the single-cell level, and observed activation of this promoter even in cells with the lowest transfection efficiency. These results show that bioluminescence imaging of single cells is a powerful tool for the analysis of gene expression based on a reporter assay using limited samples such as clinical specimens or cells from primary culture, and could provide additional information compared with the conventional assay.
authors.affiliation: Department of Pharmacoepidemiology, Graduate School of Medicine and Public Health, Kyoto University, Kyoto, Japan.
authors.forename: Tomohisa
authors.initials: T
authors.lastname: Horibe
authors.forename: Aya
authors.initials: A
authors.lastname: Torisawa
authors.forename: Ryutaro
authors.initials: R
authors.lastname: Akiyoshi
authors.forename: Yoko
authors.initials: Y
authors.lastname: Hatta-Ohashi
authors.forename: Hirobumi
authors.initials: H
authors.lastname: Suzuki
authors.forename: Koji
authors.initials: K
authors.lastname: Kawakami
mesh_terms: D002460:Cell Line; D045744:Cell Line, Tumor; D004247:DNA / Q000235:genetics*; D006801:Humans; D049449:Luminescence; D011401:Promoter Regions, Genetic; D014162:Transfection
chemical_list: D004247:DNA
keywords: LV200 system; bioluminescence; luciferase reporter assay; single-cell imaging; transfection efficiency