Page 153 - Academic Press Encyclopedia of Physical Science and Technology 3rd BioTechnology

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Encyclopedia of Physical Science and Technology EN009G-399 July 6, 2001 20:4

38 Mammalian Cell Culture

FIGURE 3 DHFR-mediated gene amplification: Cell populations containing a functional DHFR gene are exposed to
stepwise elevated concentrations of methotrexate (MTX) in the culture medium for 2 to 4 weeks at each concentration.
The majority of cells will die, but a number of clonal cells will multiply and form a monolayer that can then be exposed
to a higher level of MTX. The majority of clones subsequently analyzed will have amplified the chromosomal DNA
segments containing the DHFR gene and associated gene of interest sequences.

produce the desired protein of interest. One method for require purified protein quantities in the milligram to the
DNAtransferthathasbeenusedforthispurposeforalmost hundreds of milligram range, even before clinical studies
30 years is the calcium phosphate DNA co-precipitation are initiated. One of the limitations in large-scale transient
technique. Several improvements of the technique have expression when considering a scale beyond 20 L is the
made it very reliable and easy to use. A number of com- quantity of DNA. With an optimized calcium phosphate
mercial transfection kits are now available, most of them method, about 1 mg of plasmid DNA is required to trans-
based on synthetic or semisynthetic polymers, that work fect cells in suspension cultivated at the 1-L scale. With
very well. Usually, within a few days upon transfection, this technique, expression levels of up to 20 mg/L of a
the majority of cells have degraded or otherwise lost the recombinant antibody have been observed. One can ex-
transfected DNA. However, during this period, some DNA pect that improvements in understanding of gene transfer
sequences provided by the transfection, will have been uti- and technological breakthroughs will allow production of
lized by nuclear polymerases for transcription, producing recombinant proteins at the 100-L scale or larger in the
mRNAandthusinitiatingproteinsynthesis.Thisapproach near future.
is widely used in the setting of a standard research labo-
ratory, usually with the goal to study the function of the
transfected DNA within the cell. It has become a standard III. THE CULTURE SYSTEM
approach for the rapid synthesis and subsequent analysis
upon purification of a desired protein. With adherent cul- A. Reactors for Anchorage-Dependent Cells
tures, microgram quantities of recombinant protein can be
1. Basic Culture Units
produced quite easily. Efforts have been initiated more re-
cently to perform transient gene expression at the bioreac- Tissue culture flasks and tubes (glass or polystyrene plas-
tor scale, since many studies, especially when the product tic with special surface treatments) with surface areas
2
is eventually intended for a pharmaceutical application, of 5 to 200 cm are familiar stock items of any culture