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Nucleic Acids Research, 1985, Vol. 13, No. 12 4417-4429
© 1985

Articles

Molecular cloning of mouse tumour necrosis factor cDNA and its eukaryotic expression

Lucie Fransen, Rita Muller, Anne Marmenout, Jan Tavernier, José Van der Heyden, Eric Kawashima1, André Chollet1, Richard Tizard2, Hugo Van Heuverswyn, Adri Van Vliet, Marie-Rose Ruysschaert and Walter Fiers3

1Biogen S.A., 46, Route des Acacias, CH-1227 Carouge/Geneva, Switzerland 2Biogen Inc., 14 Cambridge Center, Cambridge, MA 02142, USA 3Laboratory of Molecular Biology Ledeganckstraat 35, B-9000 Ghent, Belgium Biogent, Jozef Plateaustraat 22, B-9000 Ghent, Belgium

Received May 7, 1985. Accepted May 31, 1985.

Tumour necrosis factor (TNF), released by induced macrophages, causes tumour necrosis in animals and kills preferentially transformed cells in vitro. mRNA induced in the established mouse monocytic PU 5.1.8 cell line by lipopolysaccharide, was converted into double-stranded cDNA and cloned in the pAT153 vector. Recombinant plasmids were screened by plus-minus hybridization and TNF-specific oligonucleotide probes constructed on the basis of partial amino acid sequences of rabbit TNF. A series of TNF specific clones were identified and confirmed by hybrid selection of mouse TNF-specific mRNA. The sequence codes for a 235 amino acids long polypeptide, of which 156 amino acids presumably correspond to the mature product. It can be concluded that mature mouse TNF is a glycosylated dimer. Biologically active TNF was secreted by both Cos-I and CHO-cells transfected with the chimaeric expression vector pSV2d2-mTNF containing the coding region of the mouse TNF cDNA gene


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