title: An in vitro amplification approach for the expression of recombinant proteins in mammalian cells.
abstract: A method for the expression of recombinant DNA products in mammalian cells based on in vitro amplification of gene units is described. Gene cassettes containing either a selectable marker or the gene of interest 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 amplified gene unit are obtained. For a first characterization of the system, the gene coding for chloramphenicol acetyltransferase (CAT) has been used to produce polymers containing a single neomycin-resistance gene ligated to different numbers of CAT gene units and used for transfection into HeLa cells. All isolated G418 (gentamycin)-resistant cell transformants which received in vitro amplified DNA were found to express high levels of CAT activity in a stable manner. Southern-blot analysis of individual clones revealed multiple copies of the gene integrated head-to-tail in the genome. This system allowed us to express the gene coding for human prepro-endothelin-1 in A617 human vascular-smooth-muscle cells. The recombinant protein was shown to be correctly processed and biologically active endothelin-1.
authors.affiliation: Department of Biological and Technological Research, San Raffaele Scientific Institute, Milan, Italy.
authors.forename: L
authors.initials: L
authors.lastname: Monaco
authors.forename: R
authors.initials: R
authors.lastname: Tagliabue
authors.forename: M R
authors.initials: MR
authors.lastname: Soria
authors.forename: M
authors.initials: M
authors.lastname: Uhlèn
mesh_terms: D001483:Base Sequence; D001704:Biopolymers; D015139:Blotting, Southern; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000235:genetics*; D002999:Clone Cells; D019332:Endothelin-1; D016232:Endothelins / Q000096:biosynthesis* / Q000378:metabolism; D015894:Genome, Human; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism; D021141:Nucleic Acid Amplification Techniques; D011498:Protein Precursors / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis; D014162:Transfection
chemical_list: D001704:Biopolymers; D019332:Endothelin-1; D016232:Endothelins; D011498:Protein Precursors; D011994:Recombinant Proteins; D015500:Chloramphenicol O-Acetyltransferase