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Abstract

Gene amplification is widely used for the production of pharmaceuticals and therapeutics in situations where a mammalian system is essential to synthesise a fully active product. Current gene amplification systems require multiple rounds of selection, often with high concentrations of toxic chemicals, to achieve the highest levels of gene amplification. The use of these systems has not been demonstrated in specialised mammalian cells, such as embryonic-stem cells, which can be used to generate transgenic animals. Thus, it has not yet proved possible to produce transgenic animals containing amplified copies of a gene of interest, with the potential to synthesise large amounts of a valuable gene product. We have developed a new amplification system, based around vectors encoding a partially disabled hypoxanthine phosphoribosyltransferase (HPRT) minigene, which can achieve greater than 1000-fold amplification of HPRT and the human growth hormone gene in a single step in Chinese hamster-lung cells. The amplification system also works in mouse embryonic-stem cells and we have used it to produce mice which express 30-fold higher levels of human protein C in milk than obtained with conventional transgenesis using the same protein C construct. This system should also be applicable to large animal transgenics produced by nuclear transfer from cultured cell lines.

Better and faster: improvements and optimization for mammalian recombinant protein production

“…Another system, called OSCAR ™ , uses a cell line that is deficient for purine synthesis. Cells are transfected with a rescue vector that supplies the gene of interest and a partially disabled hypoxanthine phosporibosyltransferase gene thus enabling selection for cell survival and amplification of the gene of interest in a single step [47]. Upon transformation and selection, a polyclonal pool of cells is established and further selection to a pure clonal line may be desirable to tease out highest producing cell lines.…”