Transfection of the ChloramphenicolAcetyltransferase Gene into Eukaryotic Cells Using Diethyl-Aminoethyl (DEAE)-Dextran
互联网
633
The study of gene regulation in eukaryotic cells involves a practical requirement for two distinct techniques: first, a transfection system, or more simply, a way of getting DNA into a cell; and second, a reporter system, which, as the name suggests, is a means of finding out what the transfected DNA does from its new location inside the cell. These two requirements are amply met by transfecting cells, in the presence of the polycation diethyl aminoethyl-dextran (DEAE-dextran), with a plasmid vector that has regulatory sequences adjacent to the chloramphenicol acetyl transferase (CAT) gene. This type of gene transfer is often referred to as a transient expression system. Production of the CAT enzyme peaks at around 40–48 h and thereafter the level falls as the plasmid DNA is diluted out in a growing population of cells. The general strategy of subcloning putative regulatory regions followed by transfection and quantification of CAT activity is outlined in a flow diagram (Fig. 1 )
Fig. 1. Flow diagram showing the key steps in transient transfection analysis. (1) Putative regulatory regions are excised from genomic DNA by restriction enzyme digestion. Fragments are (2) subcloned into the CAT vector and (3) transfected into eukaryotic cells using DEAE dextran. (4) About 40 h after transfection, the cells are lysed and the lysates incubated with chloramphenicol and radiolabeled acetyl coenzyme A. (5) Radiolabeled chloramphenicol is extracted from the mixture and (6) the level of radioactivity related to the function of the subcloned fragment.
