AN IN-VITRO AMPLIFICATION APPROACH FOR THE EXPRESSION OF RECOMBINANT PROTEINS IN MAMMALIAN-CELLS

A method for the expression of recombinant DNA products in mammalian c ells based on in vitro amplification of gene units is described. Gene cassettes containing either a selectable marker or the gene of interes t are mixed at different molar ratios, and linear polymers are formed using forced head-to-tail ligation. After introduction of the polymers into mammalian cells, transformants with various amounts of the ampli fied gene unit are obtained. For a first characterization of the syste m, the gene coding for chloramphenicol acetyltransferase (CAT) has bee n used to produce polymers containing a single neomycin-resistance gen e ligated to different numbers of CAT gene units and used for transfec tion into HeLa cells. All isolated G418 (gentamycin)-resistant cell tr ansformants which received in vitro amplified DNA were found to expres s high levels of CAT activity in a stable manner. Southern-blot analys is of individual clones revealed multiple copies of the gene integrate d head-to-tail in the genome. This system allowed us to express the ge ne coding for human prepro-endothelin-1 in A617 human vascular-smooth- muscle cells. The recombinant protein was shown to be correctly proces sed and biologically active endothelin-1.