Reddy S V, Takahashi S, Haipek C, Chirgwin J M, Roodman G D
Department of Medicine/Hematology, University of Texas Health Science Center, San Antonio.
Biotechniques. 1993 Sep;15(3):444-7.
The efficiency of DNA transfection into mammalian cell cultures has been monitored using a variety of reporter assays. However, the common procedures are expensive, time-consuming and usually cannot identify the transfected cell population directly. In the present communication we describe a simple, inexpensive and efficient method to directly identify DNA transfection in mammalian cells using tartrate-resistant acid phosphatase (TRAP) gene expression. The method involves the transfection of a plasmid (pCT3), which contains TRAP cDNA driven by a CMV promoter, into mammalian cells. The cells can then be stained for TRAP activity, and the transfection efficiency can be determined by simply counting the positively transfected cells in a defined area with a microscope. This method permits screening of mammalian cells for transfection efficiency in multi-well plates. After waiting 30-40 minutes to allow the TRAP assay to saturate, wells can be scored in 1-2 minutes with little difficulty in detecting the transfected cells.
利用多种报告基因检测方法监测了DNA转染哺乳动物细胞培养物的效率。然而，常用的方法昂贵、耗时，且通常不能直接鉴定转染的细胞群体。在本通讯中，我们描述了一种简单、廉价且高效的方法，可利用抗酒石酸酸性磷酸酶（TRAP）基因表达直接鉴定哺乳动物细胞中的DNA转染。该方法包括将一个质粒（pCT3）转染到哺乳动物细胞中，该质粒含有由CMV启动子驱动的TRAP cDNA。然后可以对细胞进行TRAP活性染色，通过在显微镜下简单计数限定区域内阳性转染细胞来确定转染效率。这种方法允许在多孔板中筛选哺乳动物细胞的转染效率。等待30 - 40分钟使TRAP检测达到饱和后，在1 - 2分钟内即可对孔进行评分，检测转染细胞几乎没有困难。
