An improved chloramphenicol acetyltransferase assay for Plasmodium falciparum transfection
An improved chloramphenicol acetyltransferase assay for Plasmodium falciparum transfection
复制标题
DOI：
10.1016/j.molbiopara.2004.03.017
复制
发表时间：
2004-08-01
影响因子：
1.5
通讯作者：
Holder, AA
中科院分区：
文献类型：
--
作者：
Lucas, SJ;Holder, AA
氯霉素乙酰转移酶（Chloramphenicol acetyltransferase， CAT）是许多不同生物（包括恶性疟原虫）转基因研究中普遍选择的报告基因。对于转染效率和基因表达的实验研究，可靠的定量分析具有重要价值。经调查发现，已发表的恶性疟原虫CAT检测方案由于孵育时间长，容易饱和；此外，从寄生虫中提取的细胞物质增加了酶的活性。开发了一种新的方案，该方案在CAT活性的三个数量级范围内定量，考虑到细胞提取效果，并且比既定方法更快。通过分析寄生虫血症和质粒数量对转染效率的影响，证明了这些改进的价值。(C) 2004 Elsevier B.V.版权所有
Chloramphenicol acetyltransferase (CAT) is a popular choice as a reporter gene in transgenic studies in many different organisms, including Plasmodium falciparum. For experimental investigations into transfection efficiency and gene expression a robustly quantitative assay is of great value. On investigation the published protocol for CAT assay of P. falciparum was found to be prone to saturation due to the long incubation time; moreover, cellular material extracted from the parasite increased the enzyme activity. A new protocol was developed which is quantitative across a range of three orders of magnitude of CAT activity, takes account of the cellular extract effect, and is more rapid than the established method. The value of these improvements was demonstrated by analysing the effects of parasitaemia and amount of plasmid on transfection efficiency with both old and new methods. (C) 2004 Elsevier B.V. All rights reserved.