AN IMPROVED CAT ASSAY FOR PROMOTER ANALYSIS IN EITHER TRANSGENIC MICE OR TISSUE-CULTURE CELLS
AN IMPROVED CAT ASSAY FOR PROMOTER ANALYSIS IN EITHER TRANSGENIC MICE OR TISSUE-CULTURE CELLS
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DOI：
10.1089/dna.1992.11.83
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发表时间：
1992-01-01
影响因子：
3.1
通讯作者：
GUERIN, SL
中科院分区：
文献类型：
--
作者：
POTHIER, F;OUELLET, M;GUERIN, SL
我们已经开发了一种改进的方法，用于确定CAT活性的指导下，稳定（转基因小鼠）或瞬时（组织培养细胞系）引入CAT报告基因构建体。该方法基于使用新的缓冲系统，该缓冲系统在制备粗细胞提取物期间显著增加了CAT酶的稳定性。当与其他程序相比，我们的方法能够增加高达100倍的检测灵敏度，这取决于测试的转基因组织。此外，在建立的组织培养细胞系中瞬时转染的各种启动子/CAT构建体也观察到强烈的增加（高达23倍）。当研究强启动子时，灵敏度的提高显着减少了进行CAT测定所需的时间（从18小时减少到1小时），并且对于分析弱启动子驱动的CAT基因表达也非常有用。
We have developed an improved method for determining CAT activity directed by stably (transgenic mice) or transiently (tissue culture cell lines) introduced CAT reporter gene constructs. The procedure is based on the use of a new buffer system which considerably increases the stability of the CAT enzyme during the preparation of the crude cell extracts. When compared to other procedures, our method enables an increase of up to 100-fold in the sensitivity of the assay, depending on the transgenic tissue tested. Furthermore, a strong increase (up to 23-fold) was also observed with various promoter/CAT constructs transiently transfected in established tissue culture cell lines. This increase in sensitivity provides a significant reduction in the time required to perform the CAT assay when strong promoters are studied (from 18 to 1 hr) and is also very useful for the analysis of CAT gene expression driven by weak promoters.