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An in vitro amplification approach for the expression of recombinant proteins in mammalian cells
Author(s) -
Monaco L.,
Tagliabue R.,
MR Soria,
Uhlen M.
Publication year - 1994
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1111/j.1470-8744.1994.tb00311.x
Subject(s) - microbiology and biotechnology , chloramphenicol acetyltransferase , recombinant dna , biology , gene , transfection , gene expression , hela , dna , in vitro , plasmid , selectable marker , cell culture , complementary dna , reporter gene , biochemistry , genetics
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.