ABSTRACT
DNA recombination and gene transfer technologies have been well established and are widely utilized in mammalian cells and animals to address a vast spectrum of biological questions. To gain an insight into the function of the gene of interest, we and others use the gene overexpression approach to up-regulate the expression of a speci„c gene followed by analysis of the potential roles of the gene in cell or animal systems.1-5 Gene overexpression refers to an increase in the amount of a given protein or the product level of a gene, which may result in an alteration in the function of the gene. We have successfully transfected mammalian cells with DNA constructs containing sense HSP27 cDNA and developed stably transfected cell lines in which the level of HSP27 increased by 4-to 8-fold as compared with nontransfected cells. As a result, cells containing higher levels of HSP27 can protect the cells against heat and toxicants such as heavy metals, demonstrating that one of the functions of the HSP27 protein is to enable cells to survive and recover from stress conditions.1 In addition, gene overexpression has a broad range of applications in animal systems.2-6 It is also applied to treat some human diseases resulting from defects in single genes by transferring the cDNA of a normal gene into patients to compensate for the lack of a given protein, which is termed gene therapy. Therefore, gene overexpression technology constitutes one of the major advances in current medicine and molecular genetics.3,6,7-11
How to overexpress a gene of interest? The general procedures are outlined in Figure 8.1. The primary principles are that the sense cDNA constructs of interest are „rst introduced into cells and integrated into the chromosomal DNA of the host, and that the expression of exogenous cDNA is driven by an appropriate promoter to produce sense RNA that is translated into protein. In this way, the endogenous protein plus the exogenous protein signi„cantly elevate the level of the protein in the cell or animal. There are two types of transfection and expression in mammalian cells. One is transient transfection in which exogenous cDNA is introduced into cells, and the cDNA is allowed to be expressed for 1 to 3 days. The transfected cells are lysed and the proteins are analyzed. The purpose of transient transfection is usually to obtain a burst in the expression of the transferred cDNA. However, such transfection is not suitable for the selection of stably transfected cell lines. The other is stable transfection in which foreign genes are introduced into cells and are stably integrated into the chromosomes or genomes of the host cells. The integrated DNA can replicate ef„ciently and is maintained during cell division. The expressed products can
be analyzed from successive generations of divided cells, establishing genetically altered cell lines.
