A recombinant Chinese hamster ovary cell line containing a 300-fold amplified tetramer of the hepatitis B genome together with a double selection marker expresses high levels of viral protein.
A new series of double-selection plasmids containing recombinant genes expressing the neomycin phosphotransferase (NEO) of transposon Tn5 and mouse dihydrofolate reductase (DHFR) in mammalian cells is described. Activity of the recombinant DHFR gene varied more than 50-fold, depending on the location of the simian virus 40 72 base-pair repeat or enhancer, which is part of the promoter of the NEO unit. A NEO-DHFR module with the enhancer located at the 3' end of the DHFR gene was inserted into a plasmid containing four tandem head-to-tail copies of the hepatitis B virus (HBV) genome and the new plasmid was used to transform DHFR- Chinese hamster ovary cells. In one of the cell lines obtained, an unrearranged copy of the HBV tetramer could be amplified 300-fold by increasing selective pressure with methotrexate, resulting in a proportional increase of the synthesis of HBV surface antigen. Four different mRNAs detected in the amplified cell line probably encode HBV core protein, pre-S and surface antigens, and the X protein. As a result of the DNA amplification, synthesis of HBV proteins is no longer restricted to resting cells. Integrated plasmid sequences appear to be stable during the amplification process.
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004274:DNA, Recombinant; D005260:Female; D005784:Gene Amplification; D005814:Genes, Viral; D005819:Genetic Markers; D006511:Hepatitis B Antigens / Q000096:biosynthesis; D006515:Hepatitis B virus / Q000235:genetics*; D019868:Kanamycin Kinase; D010053:Ovary / Q000166:cytology; D010770:Phosphotransferases / Q000235:genetics; D010957:Plasmids; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics; D014764:Viral Proteins / Q000096:biosynthesis
D004274:DNA, Recombinant; D005819:Genetic Markers; D006511:Hepatitis B Antigens; D014764:Viral Proteins; D013762:Tetrahydrofolate Dehydrogenase; D010770:Phosphotransferases; D019868:Kanamycin Kinase