Cloning of genomic DNA for tumor necrosis factor and efficient expression in CHO cells.
A genomic clone for human tumor necrosis factor (TNF-alpha) was isolated using synthetic oligonucleotide probes. The genomic DNA was cleaved to remove 5' regulatory sequences and cloned in a PSVE3 expression vector containing the SV40 early promoter. The plasmid was co-transfected with a selectable dihydrofolate reductase (DHFR) gene into DHFR-deficient Chinese hamster ovary cells. Efficient expression of TNF mRNA was established by Northern analysis. Expression of TNF protein was assayed for by cytotoxic activity for cycloheximide-treated SV80 fibroblasts. Selected transfected cultures secreted as much as 50,000 units of TNF activity/ml of culture medium. Synthesis of TNF protein was confirmed by immunofluorescence of transfected cells with a monoclonal antibody to TNF and immunoprecipitation of 17 kD protein from transfected CHO culture supernates. The efficient expression of TNF from genomic DNA in transfected mammalian cells may be advantageous for biologic uses.
Korn
J H
JH
Department of Virology, Weizmann Institute for Science, Rehovot, Israel.
Mory
Y
Y
Ziberstein
A
A
Holtmann
H
H
Revel
M
M
Wallach
D
D
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D004247:DNA / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics*
D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D004247:DNA