RRC ID 47470
著者 Endo T, Noda N, Kuromi Y, Kokura K, Kazuki Y, Oshimura M, Ohbayashi T.
タイトル Evaluation of an Hprt-Luciferase Reporter Gene on a Mammalian Artificial Chromosome in Response to Cytotoxicity.
ジャーナル Yonago Acta Med
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.
巻・号 59(2)
ページ 174-82
公開日 2016-6-1
PMID 27493490
PMC PMC4973025
IF 0.974
引用数 4
WOS 分野 MEDICINE, RESEARCH & EXPERIMENTAL
リソース情報
遺伝子材料 B6N Mouse BAC (RDB07573) B6Ng01-126E09