Selection of Highly Productive Mammalian Cells Based on an Inducible Growth Advantage Using an Antibody/Receptor Chimera
- نوع فایل : کتاب
- زبان : انگلیسی
- مؤلف : MASAHIRO KAWAHARA, I* HIROSHI UEDA,12 KOUHEI TSUMOTO, 3 IZUMI KUMAGAI, 3 WALT MAHONEY, 4 AND TERUYUKI NAGAMUNE 1
- چاپ و سال / کشور: 2002
Description
In mammalian cell culture, the selection of high producers is a critical step in efficient recombinant protein production. Drug-resistance selection has been commonly used, but does not always give a pure population of high producers. In this study, we propose a novel selection method in which the growth of high producers is specifically promoted. Two plasmids encoding (i) a hybrid receptor composed of the V H portion of anti-hen egg lysozyme antibody HyHEL-10 and an N-terminally truncated erythropoietin receptor (Va-EpoR), and (ii) a VL-EpoR fusion derived from the same construct as in (i), were employed. The second plasmid contained enhanced green fluorescent protein (EGFP) as a modei recombinant protein that was flanked by the internal ribosomal entry sequence. Both plasmids were used simultaneously to transfect an IL-3-dependent murine myeloid cell line, 32D. The transfectants, after antigen selection in the absence of IL-3, showed a clear antigen-induced dose-dependent proliferation. In addition, a high EGFP expression level was observed by flow cytometry in comparison with the cells before antigen selection. The results clearly demonstrate the advantage of our method over conventional drug-resistance selection. We propose the term AMEGA (Antigen MEdiated Genetically-modified cell Amplification) for such an approach.
JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 93, No. 4, 399404. 2002 Received 12 November 2001/Accepted 21 January 2002
