Evaluation of an Hprt-Luciferase Reporter Gene on a Mammalian Artificial Chromosome in Response to Cytotoxicity
Evaluation of an Hprt-Luciferase Reporter Gene on a Mammalian Artificial Chromosome in Response to Cytotoxicity
复制标题
哺乳动物人工染色体上 Hprt-荧光素酶报告基因对细胞毒性反应的评估
作者：
Takeshi Endo;N. Noda;Yasushi Kuromi;K. Kokura;Y. Kazuki;M. Oshimura;T. Ohbayashi
背景
次黄嘌呤鸟嘌呤磷酸核糖基转移酶（Hprt）被认为是管家基因，并已被用作实时定量RT-PCR和各种其他基因表达分析方法的内部对照。为了评估Hprt mRNA水平作为参考标准，我们设计了由长Hprt启动子驱动的荧光素酶报告基因，并测量了其对细胞毒性的反应。
方法
我们构建了一个报告载体，该载体含有一个phiC 31整合酶识别位点和一个小鼠Hprt启动子，并与绿色荧光素酶（SLG）编码序列融合。在小鼠A9细胞中使用phiC 31整合酶将Hprt-SLG载体加载到含有多整合酶平台的小鼠人工染色体上。我们建立了三个独立的克隆体。
结果
建立的细胞系具有相似的Hprt-SLG报告基因表达水平。Hprt-SLG活性在生长条件下成比例地增加，并且在杀稻瘟菌素或顺铂施用后在细胞毒性条件下降低。在生长和细胞毒性条件下分别观察到SLG发光的类似增加和减少，与使用市售试剂alamarBlue获得的荧光中的那些类似。
结论
通过在哺乳动物人工染色体中采用可靠和稳定的表达系统，Hprt-SLG报告基因的活性可以反映细胞生长条件下的细胞数量和细胞毒性条件评价中的细胞活力。
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.
影响因子：
3.5
作者：
Olivares, EC;Hollis, RP;Calos, MP
通讯作者：
Calos, MP