Threonine synthesis from homoserine as a selectable marker in mammalian cells

Abstract

The plasmid pSVthrBC expresses the Escherichia coli thrB (homoserine kinase) and thrC (threonine synthase) genes in mouse cells and enables them to synthesize threonine from homoserine. After transfection with pSVthrBC and culture in medium containing homoserine, only cells that have incorporated pSVthrBC survive. Homoserine at concentrations greater than 1 mM is toxic to mammalian cells. Mouse cells selected from medium containing 5 mM homoserine had incorporated 20-100 copies of the plasmid per cell and had homoserine kinase activities of 0.001-0.012 nmol/min per mg of protein per copy. Cells selected from medium containing 10 mM homoserine had incorporated one or two copies of the plasmid per cell and had homoserine kinase activities of 0.06-0.39 nmol/min per mg of protein per copy. By using high concentrations of homoserine, it is possible to use pSVthrBC to select and isolate cell lines that have one or two copies of the plasmid incorporated into an active region of chromatin. CHO and HeLa cells have also been successfully transfected with pSVthrBC. COS-7 cells are naturally resistant to homoserine as they are able to metabolize homoserine.