A one-step gene amplification system for use in cultured mammalian cells and transgenic animals

Gene amplification is widely used for the production of pharmaceuticals and therapeutics in situations where a mammalian system is essential to synthe sise a fully active product. Current gene amplification systems require mul tiple rounds of selection, often with high concentrations of toxic chemical s, to achieve the highest levels of gene amplification. The use of these sy stems has not been demonstrated in specialised mammalian cells, such as emb ryonic-stem cells, which can be used to generate transgenic animals. Thus, it has not yet proved possible to produce transgenic animals containing amp lified 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 pho sphoribosyltransferase (HPRT) minigene, which can achieve greater than 1000 -fold amplification of HPRT and the human growth hormone gene in a single s tep in Chinese hamster-lung cells. The amplification system also works in m ouse 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 conven tional 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.