High-Level Expression in Cells

Proteins or enzymes of interest can be expressed at very high levels, which can be advantageous for studying certain functional alterations. The most commonly used systems include plasmid-transformed bacteria (Oudenampsen et al. 1990), baculovirus-infected insect cells (Kost and Condreay 1999), and vaccinia-virus-infected mammalian cells (Chakrabarti et al. 1985; Eckert and Merchlinsky 1999). All three systems allow high-level expression of cloned genes. In general, bacterial expression is the easiest to use. However, both the baculovirus-infected and the vaccinia-virus-infected expression systems are eukaryotic, and it might be particularly important to use them if post-translational processing or intracellular accessory factors are needed in the production of a functional gene product. Moreover, proteins expressed in any of the above systems can be given “tag sequences” to allow for rapid, specific purification.

Transient and Stable Transfections of Mammalian Cells in Culture

Yeast (Oeda et al. 1985), African green monkey kidney fibroblast COS cells (Zuber et al.1986), and vaccinia virus (Battula et al. 1987) were among the earliest expression systems in vivo. They have been successfully used to study DMEs. Allelic DNA can be transfected (passed into the cell) by microinjection or by chemical-DNA aggregation methods including calcium phosphate precipitation and liposome-mediated transfection. By using such methods, followed by antibiotic treatment to isolate cells that house the plasmid containing the selectable marker gene, cells can be transfected either transiently or stably. In transient transfections, expression of the gene is generated from extrachromosomal copies of the transfected plasmid and persists until the expression plasmid is degraded or diluted by cell passage. In general, 5% to 50% of all cells in culture contain the incoming gene, the DNA is not stably “integrated” in the cell’s genome, and the transfected cells contain many copies of the new genetic material. In contrast, for stable transfections, the incoming DNA is integrated (albeit randomly) into the cell’s genome. Cells expressing the gene under study are initially selected on the basis of co-expression of a gene that provides antibiotic resistance. After antibiotic selection, continued cellular propagation in the presence of the antibiotic will ensure that the gene of interest is expressed in a more or less permanent (i.e., stable) fashion—remaining after the cells are passaged on through many additional generations. The copy number of integrated genes is highly variable and can range from one to several dozen, and the genes are generally arranged in tandem (head to tail) at an “integration site” that normally cannot be directed, or controlled for, on an experimental basis.

For better gene expression in culture, one or more introns (even when they are artificially inserted) have been found to enhance gene activity in cultured cells (Palmiter et al. 1991); alternatively, small genes with fewer than 10 and



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