When exposed to cytotoxic drugs, some cells can develop a drug-resistant pheno-type. Although there are mechanisms to generate drug tolerance that do not employ gene amplification, amplification of DNA in response to growth-limiting conditions is a well-known phenomenon (51), and one that can be exploited in order to maximize expression of recombinant products. In this case, the gene that is targeted for amplification is the transfected marker gene. During the process of amplification, both the target gene and surrounding DNA sequences are coampli-fied via a process that may involve over-replication of DNA and/or recombination of sequences. As a result, amplification of the marker gene provides an indirect means of amplifying the cointegrated product gene(s) (50,51,76-78). MTX irreversibly binds to and inhibits the activity of DHFR, therefore dose-dependently limiting the cells ability to produce tetrahydrofolate. Under conditions in which the MTX concentration is limiting for growth, amplification of the DHFR gene frequently occurs. This effect has been exploited in the development of highly productive commercial cell lines. A similar effect can be obtained using the GS selection marker and MSX. The DNA replicated during the isolation of a drug-resistant phenotype is structurally referred to as an amplicon, and can contain a few to thousands of copies of the amplified gene (79). Increases in the gene copy number of a given cell line are often associated with increased product expression or secretion. However, since not all gene copies may be transcriptionally active or other bottlenecks to protein expression may occur, cell lines with high gene copy numbers may not always be highly productive (79).
The initial selection of the transfected cells in selective media or in low concentrations of the selective agent is generally thought to identify cells which have either integrated multiple copies of the plasmid or which have single or low copy number integrations at exceptionally active transcription loci. The more stringent the selection, the higher the initial expression level of the cell pool and the more frequently high expressors will occur within this pool. In cases where amplification is desired, these initial selectants are then exposed to stepwise increases in the toxic agent. The steepness of the increase in selective agent during gene amplification protocols seems to have an impact on whether drug-resistant cells have increased copy number of the integrated sequences and co-express high levels of drug product or have acquired drug resistance through mechanisms not involving gene amplification (80). Detailed analyses of integration site, gene copy number, and specific productivity of clones resulting from various types of selection and amplification strategies are in progress to better define the types of events which lead to highly productive, stable cell lines (81,82). However, at present, the optimal selection and amplification procedures appear to be highly dependent on the design of the expression construct and needs to be determined independently for each system.
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