Takeshi Endo1, Natsumi Noda1, Yasushi Kuromi2, Kenji Kokura3, Yasuhiro Kazuki4, Mitsuo Oshimura3, Tetsuya Ohbayashi5. 1. Tottori Industrial Promotion Organization, Tottori 689-1112, Japan. 2. Tottori Industrial Promotion Organization, Tottori 689-1112, Japan; ‡Division of Laboratory Animal Science, Research Center for Bioscience and Technology, Tottori University, Yonago 683-8503, Japan. 3. §Chromosome Engineering Research Center, Tottori University, Yonago 683-8503, Japan. 4. §Chromosome Engineering Research Center, Tottori University, Yonago 683-8503, Japan; ||Department of Biomedical Science, Institute of Regenerative Medicine and Biofunction, Tottori University, Yonago 683-8503, Japan; ¶Division of Molecular and Cell Genetics, Department of Molecular and Cellular Biology, School of Life Sciences, Tottori University Faculty of Medicine, Yonago 683-8503, Japan. 5. ‡Division of Laboratory Animal Science, Research Center for Bioscience and Technology, Tottori University, Yonago 683-8503, Japan.
Abstract
BACKGROUND: Hypoxanthine guanine phosphoribosyltransferase (Hprt) is known as a house-keeping gene, and has been used as an internal control for real-time quantitative RT-PCR and various other methods of gene expression analysis. To evaluate the Hprt mRNA levels as a reference standard, we engineered a luciferase reporter driven by a long Hprt promoter and measured its response to cytotoxicity. METHODS: We constructed a reporter vector that harbored a phiC31 integrase recognition site and a mouse Hprt promoter fused with green-emitting luciferase (SLG) coding sequence. The Hprt-SLG vector was loaded onto a mouse artificial chromosome containing a multi-integrase platform using phiC31 integrase in mouse A9 cells. We established three independent clones. RESULTS: The established cell lines had similar levels of expression of the Hprt-SLG reporter gene. Hprt-SLG activity increased proportionately under growth conditions and decreased under cytotoxic conditions after blasticidin or cisplatin administration. Similar increases and decreases in the SLG luminescent were observed under growth and cytotoxic conditions, respectively, to those in the fluorescent obtained using the commercially available reagent, alamarBlue. CONCLUSION: By employing a reliable and stable expression system in a mammalian artificial chromosome, the activity of an Hprt-SLG reporter can reflect cell numbers under cell growth condition and cell viability in the evaluation of cytotoxic conditions.
BACKGROUND:Hypoxanthine guanine phosphoribosyltransferase (Hprt) is known as a house-keeping gene, and has been used as an internal control for real-time quantitative RT-PCR and various other methods of gene expression analysis. To evaluate the Hprt mRNA levels as a reference standard, we engineered a luciferase reporter driven by a long Hprt promoter and measured its response to cytotoxicity. METHODS: We constructed a reporter vector that harbored a phiC31 integrase recognition site and a mouseHprt promoter fused with green-emitting luciferase (SLG) coding sequence. The Hprt-SLG vector was loaded onto a mouse artificial chromosome containing a multi-integrase platform using phiC31 integrase in mouse A9 cells. We established three independent clones. RESULTS: The established cell lines had similar levels of expression of the Hprt-SLG reporter gene. Hprt-SLG activity increased proportionately under growth conditions and decreased under cytotoxic conditions after blasticidin or cisplatin administration. Similar increases and decreases in the SLG luminescent were observed under growth and cytotoxic conditions, respectively, to those in the fluorescent obtained using the commercially available reagent, alamarBlue. CONCLUSION: By employing a reliable and stable expression system in a mammalian artificial chromosome, the activity of an Hprt-SLG reporter can reflect cell numbers under cell growth condition and cell viability in the evaluation of cytotoxic conditions.
Authors: Bosiljka Tasic; Simon Hippenmeyer; Charlene Wang; Matthew Gamboa; Hui Zong; Yanru Chen-Tsai; Liqun Luo Journal: Proc Natl Acad Sci U S A Date: 2011-04-04 Impact factor: 11.205