Transfection of murine myeloma cells to produce a chimeric antibody to the interleukin-2 receptor.
Murine myeloma X63Ag8.653 cells were transfected with heavy and light-chain expression vectors for a chimeric antibody (Ab) to the human interleukin-2 receptor. A cell line producing low quantities of the chimeric Ab was obtained and was transfected with either the cytomegalovirus (CMV) immediate-early gene ie1 or Epstein-Barr virus (EBV) BMLF1 DNA, together with the hygromycin B resistance (HyR) encoding gene for selection to improve productivity. Two cell lines with a four to eightfold increase in productivity were obtained. They showed higher levels of heavy- and light-chain mRNA expression. CMV ie1 or EBV BMLF1 DNA was not detected and no integration pattern changes for the heavy- and light-chain DNA were seen. The long-term productivity of one of the cell lines showed hygromycin B (Hy) requirement. Transfection with the HyR DNA alone also resulted in cells with increased productivity. The expression vectors contained the immunoglobulin light-chain enhancer kappa B DNA sequences (kappa B site). Nuclear extracts from parent myeloma cells showed one kappa B-binding protein band on a polyacrylamide gel, but nuclear extracts from transfected cells showed two additional slower-migrating bands. Increased Ab production correlated with an increased ratio of the medium-mobility kappa B-binding protein band to the high-mobility band. The possibility that Hy used for selection activated kappa B-binding proteins and increased Ab expression is discussed.
Meng
Y G
YG
Department of Cellular and Molecular Biology Research, Miles Inc., Berkeley, CA 94701.
Wong
T
T
D000818:Animals; D000906:Antibodies / Q000235:genetics*; D001483:Base Sequence; D002460:Cell Line; D003587:Cytomegalovirus / Q000235:genetics; D004279:DNA, Viral; D004591:Electrophoresis, Polyacrylamide Gel; D017781:Genes, Immediate-Early; D004854:Herpesvirus 4, Human / Q000235:genetics; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D015375:Receptors, Interleukin-2 / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D014162:Transfection; D014407:Tumor Cells, Cultured
D000906:Antibodies; D004279:DNA, Viral; D009838:Oligodeoxyribonucleotides; D015375:Receptors, Interleukin-2; D011993:Recombinant Fusion Proteins