Endogenous gene and amplifiable cDNA construct both produce unstable t-PA mRNA in Bowes melanoma cells.
Bowes melanoma cells, which naturally produce tissue-type plasminogen activator (t-PA), were transfected with a plasmid containing a human t-PA cDNA under transcriptional control of the promoter/enhancer of the major immediate early gene of human cytomegalovirus (CMV) plus genes expressing geneticin (G418) resistance and dihydrofolate reductase (DHFR). In one of the initial geneticin-resistant transformants, t-PA mRNA transcribed from the chromosomally integrated plasmid had the same short half-life, 20-30 min, as did mRNA transcribed from the endogenous t-PA gene compared to 7-8 h for total poly(A)+ mRNA. After subsequent selection of such cells with methotrexate, a cell line was obtained in which the t-PA cDNA construct was co-amplified with the DHFR gene and which produced 10 times more t-PA protein than the original Bowes melanoma cells.
CIBA-GEIGY Ltd., Biotechnology Department, Basle, Switzerland.
F A
FA
Asselbergs
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P
van Someren
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J
van Meerwijk
D003001:Cloning, Molecular; D004351:Drug Resistance / Q000235:genetics; D005784:Gene Amplification; D006207:Half-Life; D006801:Humans; D008545:Melanoma; D008727:Methotrexate / Q000494:pharmacology; D010957:Plasmids; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism*; D010959:Tissue Plasminogen Activator / Q000235:genetics*; D014162:Transfection; D014407:Tumor Cells, Cultured
D012333:RNA, Messenger; D010959:Tissue Plasminogen Activator; D008727:Methotrexate