Amplification of expression of hepatitis B surface antigen in 3T3 cells cotransfected with a dominant-acting gene and cloned viral DNA.
3T3 cells containing hepatitis B virus DNA sequences can be efficiently selected by exposure to methotrexate after cotransfection with cloned viral DNA and DNA coding for a methotrexate-resistant dihydrofolate reductase. More than 75% of methotrexate-resistant cells isolated after cotransfection with a head-to-tail tandem of the hepatitis B virus genome synthesized viral surface antigen. The antigen was released into the culture medium in the form of 22-nm particles with buoyant density of 1.20 g/ml. No other virally coded proteins were detected in the cells or the culture medium. Application of selective pressure by increasing the concentration of methotrexate resulted in an amplification of viral DNA sequences and a concomitant increase in the rate of synthesis and release of hepatitis B surface antigen. The ability to produce large amounts of surface antigen appears to be a stable trait and has been maintained in these cultures through more than 30 passages.
D000818:Animals; D002460:Cell Line; D004351:Drug Resistance; D005784:Gene Amplification / Q000187:drug effects*; D005786:Gene Expression Regulation / Q000187:drug effects; D006514:Hepatitis B Surface Antigens / Q000096:biosynthesis / Q000235:genetics*; D006515:Hepatitis B virus / Q000235:genetics*; D008727:Methotrexate / Q000494:pharmacology; D051379:Mice; D010957:Plasmids
D006514:Hepatitis B Surface Antigens; D008727:Methotrexate