Molecular analysis of successful cell line selection in transfected GS-NS0 myeloma cells.
The production of recombinant proteins from mammalian cells is now an essential part of biotechnology. However, despite this importance, the detailed characteristics of good producing cell lines remain largely unknown. The industrially important GS-NS0 mammalian expression system is able to produce large amounts of protein from relatively few copies of recombinant genes. This makes GS-NS0 cell lines ideal candidates to study the consequence of recombinant plasmid transfection in mammalian cells. This study investigated the molecular features of a panel of 17 randomly chosen GS-NS0 cell lines engineered to produce a recombinant antibody. The research analysed antibody production via enzyme-linked immunosorbent assay (ELISA), and investigated the molecular features of the transfectants by Northern, Southern and copy number analysis. The cell lines generated produced a range of antibody concentrations. In addition, for transfectants defined as producers of recombinant antibody there was a positive correlation between specific productivity and heavy chain mRNA expression. The use of Northern and Southern analysis allowed determination of the functional integrity of the transfected plasmid. Over 50% of the transfectants studied had molecular defects at the level of mRNA and/or cDNA. Cell lines were identified with suspected defects in the regulatory regions of transfected genes in addition to cell lines which lacked recombinant genes. Also, "false-positive" cell lines were generated which were able to overcome the GS selection pressure without producing any recombinant antibody. This article discusses these findings in relation to vector design.
Barnes
Louise M
LM
Faculty of Life Sciences, The Michael Smith Building, University of Manchester, Oxford Road, Manchester M13 9PT, UK. louise.m.barnes@manchester.ac.uk
Bentley
Catherine M
CM
Moy
Nicola
N
Dickson
Alan J
AJ
D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000235:genetics; D015152:Blotting, Northern; D015139:Blotting, Southern; D002460:Cell Line; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D006801:Humans; D009101:Multiple Myeloma / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection
D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins