title: A one-step gene amplification system for use in cultured mammalian cells and transgenic animals.
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.
authors: Sir Alastair Currie CRC Laboratories, Molecular Medicine Centre, Edinburgh University, Western General Hospital, Scotland, UK. David.Melton@ed.ac.uk
authors: D W
authors: DW
authors: Melton
authors: A M
authors: AM
authors: Ketchen
authors: A J
authors: AJ
authors: Kind
authors: C
authors: C
authors: McEwan
authors: D
authors: D
authors: Paisley
authors: J
authors: J
authors: Selfridge
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics*; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D004622:Embryo, Mammalian / Q000166:cytology / Q000201:enzymology; D005784:Gene Amplification; D006168:Guinea Pigs; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D008892:Milk / Q000737:chemistry; D011486:Protein C / Q000032:analysis; D012333:RNA, Messenger / Q000235:genetics; D013234:Stem Cells / Q000201:enzymology
chemical_list: D011486:Protein C; D012333:RNA, Messenger; D007041:Hypoxanthine Phosphoribosyltransferase