title: Evaluation of an Hprt-Luciferase Reporter Gene on a Mammalian Artificial Chromosome in Response to Cytotoxicity.
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.
  
  
  CONCLUSIONS
  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: Tottori Industrial Promotion Organization, Tottori 689-1112, Japan.
authors: Takeshi
authors: T
authors: Endo
authors: Tottori Industrial Promotion Organization, Tottori 689-1112, Japan.
authors: Natsumi
authors: N
authors: Noda
authors: 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.
authors: Yasushi
authors: Y
authors: Kuromi
authors: §Chromosome Engineering Research Center, Tottori University, Yonago 683-8503, Japan.
authors: Kenji
authors: K
authors: Kokura
authors: §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.
authors: Yasuhiro
authors: Y
authors: Kazuki
authors: §Chromosome Engineering Research Center, Tottori University, Yonago 683-8503, Japan.
authors: Mitsuo
authors: M
authors: Oshimura
authors: ‡Division of Laboratory Animal Science, Research Center for Bioscience and Technology, Tottori University, Yonago 683-8503, Japan.
authors: Tetsuya
authors: T
authors: Ohbayashi
keywords: gene reporter; hypoxanthine phosphoribosyltransferase; luciferase; mouse artificial chromosome; reference standards