Harrison Ewen M, Garden O J, Ross James A, Wigmore Stephen J
Tissue Injury and Repair Group, University of Edinburgh, Chancellor's Building, Little France Crescent, UK.
J Immunol Methods. 2006 Mar 20;310(1-2):182-5. doi: 10.1016/j.jim.2005.11.022. Epub 2006 Jan 13.
Luciferase reporter constructs are an accurate method of assessing gene promoter activity and vectors constitutively expressing luciferase are useful in quantifying transfection efficiency. Common methodologies for examining the induction of the heat shock (stress) response require exposure of cells transfected with luciferase-expressing vectors to a mild heat stress. Here we re-examine the under-recognised phenomenon that luciferase is exquisitely sensitive to small temperature changes. In cells subjected to mild heat exposure following transfection with both luciferase and beta-galactosidase reporter vectors, a marked reduction in luciferase activity was observed compared with beta-galactosidase activity. On exposing recombinant firefly luciferase to small increases in temperature in vitro, a time and temperature dependent decrease in luciferase activity was demonstrated. Loss of luciferase activity following mild heat exposure will result in misinterpretation of reporter activity. This vastly underappreciated effect is worthy of further emphasis and luciferase reporter vectors should be used with caution in protocols that involve exposure to temperatures outside the physiological range.
荧光素酶报告基因构建体是评估基因启动子活性的一种准确方法，而组成型表达荧光素酶的载体可用于量化转染效率。检测热休克（应激）反应诱导的常用方法要求将转染了表达荧光素酶载体的细胞暴露于轻度热应激下。在此，我们重新审视了一个未得到充分认识的现象，即荧光素酶对微小的温度变化极为敏感。在用荧光素酶和β-半乳糖苷酶报告基因载体转染后经历轻度热暴露的细胞中，与β-半乳糖苷酶活性相比，观察到荧光素酶活性显著降低。在体外将重组萤火虫荧光素酶暴露于温度小幅升高的环境中时，荧光素酶活性呈现出时间和温度依赖性降低。轻度热暴露后荧光素酶活性的丧失将导致对报告基因活性的错误解读。这种被极大低估的效应值得进一步强调，在涉及暴露于生理范围之外温度的实验方案中，应谨慎使用荧光素酶报告基因载体。
