The initial goal of cell line development is to identify rare, stable, monoclonal cells secreting high titres of a biologically active recombinant protein such as a monoclonal antibody (mAb).
Many different strategies can be used to produce a pool of recombinant cells, with different laboratories preferring or specialising in different transfection methods. The standard approach starts with a bulk transfection (around 10-20 x 106 cells) of cells such as CHO-S, followed by clone selection, typically with methotrexate (MTX) amplification or Lonza’s glutamine synthetase (GS) system. However, the random integration of recombinant DNA is an inefficient way of generating high-producing clones. It is therefore necessary to screen many hundreds or thousands of transfected cells to isolate a clone with suitable properties.
Some newer approaches in cell line engineering such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and meganucleases have been designed to target transgene integration to specific DNA sequences in the genome. This can increase the frequency of stable high-producers and reduce the number of clones for screening.
After transfection or fusion, certain fluorescence-based methods can be used to enrich for high producers, for example:
The top candidate clones are seeded into microplates for further investigation. For more information, see cell seeding.
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