Gene transfer and gene amplification in mammalian cells

Abstract

This chapter discusses some aspects of gene transfer and amplification in dihydrofolate reductase-deficient (dhfr-deficient) Chinese Hamster Ovary (CHO) cells, one of the most popular mammalian cell hosts for recombinant protein production. The observations made and conclusion drawn have value also for work with other mammalian cell hosts for recombinant protein production. Expression vectors containing the target gene are used in combination with a functional dhfr expression cassette either on the same or on a separate vector. Few firmly established insights have been gained about the integration of transfected DNA into the genome of the host cell, but it appears that mitotic activity of the host cell population during the transfection procedure is an important parameter. Selection and identification of recombinant cells is performed using the DHFR phenotype. Integration into a chromosomal site within the nucleus is random and rare, usually guaranteeing a singular event. Recombinant cell clones might vary with respect to the stability within the primary locus of integration. Profound effects on transcription rates and on the eventual fate of transfected DNA are exerted by the genomic DNA environment at the integration site. Identification of highly productive cell lines requires screening of candidate clonal cell populations. In spite of many unanswered questions, CHO cells have been extremely successful as a reliable source of large quantities of high-value proteins. No other cell line is available that has been studied to the same extent as CHO cells with respect to a multitude of parameters and characteristics. Though many details remain to be deciphered about gene transfer and amplification in mammalian cells, further study of CHO cells surely will lead to an improved understanding of these complex mechanisms.