Andre Lieber

[18] – Stable High-Level Gene Expression in Mammalian Cells by T7 Phage RNA Polymerase

Publisher Summary This chapter provides an overview of gene expression in mammalian cells. Stable expression of foreign genes can be achieved using constructs that either integrate into the host cell genome or have the ability to replicate at a moderate level. Vectors of the second type are preferentially derived from bovine papilloma virus (BPV) and from Epstein-Barr virus. Integrating plasmid vectors can be amplified by linkage to genes such as that for dihydrofolate reductas or metallothionein and subsequent selection with increasing drug concentrations. Differences in the expression levels between individual clones derived from one and the same transfection can be enormous, ranging between only a few nanograms and several micrograms. In mammalian cells, T7 RNA polymerase gene leads to cytoplasmic localization of the polymerase. The chapter additionally describes the methods of purification of plasmids for transfection. Plasmids are purified by two rounds of cesium chloride gradient centrifugation. Superior purity is achieved by column chromatography on BD-cellulose. The chapter further discusses about synthesis of antisense RNA and ribozymes.