Pothier F, Ouellet M, Julien J P, Guérin S L
Département de Physiologie, Faculté de Médecine, Université Laval, Québec, Canada.
DNA Cell Biol. 1992 Jan-Feb;11(1):83-90. doi: 10.1089/dna.1992.11.83.
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.
我们已经开发出一种改进方法，用于测定由稳定导入（转基因小鼠）或瞬时导入（组织培养细胞系）的氯霉素乙酰转移酶（CAT）报告基因构建体所指导的CAT活性。该方法基于一种新缓冲液系统的使用，该系统在制备粗细胞提取物过程中能显著提高CAT酶的稳定性。与其他方法相比，我们的方法可使检测灵敏度提高达100倍，具体取决于所检测的转基因组织。此外，对于在已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，也观察到灵敏度大幅提高（高达23倍）。当研究强启动子时，这种灵敏度的提高显著减少了进行CAT检测所需的时间（从18小时减至1小时），并且对于分析由弱启动子驱动的CAT基因表达也非常有用。
