Page 152 - Academic Press Encyclopedia of Physical Science and Technology 3rd BioTechnology

P1: GRB/GUB P2: FYD Final Pages
Encyclopedia of Physical Science and Technology EN009G-399 July 6, 2001 20:4

Mammalian Cell Culture 37

FIGURE 2 DHFR-mediated transfection: One, two, or more plasmids containing a functional DHFR expression
cassette and other gene of interests can be (co-) transfected into Chinese hamster ovary cells that are DHFR negative.
It is preferable to linearize the DNA to be transfected. Individual cells that have taken up and express the DHFR gene
will survive and form colonies in a medium that lacks nucleotide precursor molecules [glycin, hypoxonthin, thymidin
(GHT)-minus medium].

is randomly integrated in mammalian chromosomes. The grow at elevated MTX concentrations. Over periods of
targeted integration is mediated by homologous recombi- weeks and eventually months, subclones of cell lines can
nation whereby the targeting vector will contain sequences be generated that are resistant to very high levels (up to
similar or identical to the DNA at the target site. Since 300 µM) of MTX (see Fig. 3). Such cell lines are very
targeted integration is dependent on the chance of close frequently found to have amplified segments of chromo-
physical proximity of plasmid DNA and the target site, somal regions containing the DHFR gene and the gene of
random integration will still occur at a higher frequency. interest. The result of gene amplification is not only an
To increase the frequency of targeted over non-targeted elevated expression of DHFR but also of the secondary
integration of foreign DNA in the mammalian genome, gene of interest. This increase in expression rarely cor-
selection strategies can be applied in culture that employ relates linearly with the increase in copy number, yet 5-
two different selection markers—for example, a neomycin to 20-fold improvements of the specific productivity of
resistance gene and a herpes simplex thymidine kinase stable cell lines have been found.
gene, both sequences adjacent to 10 to 15 kilobases of
homologous DNA.
4. Transient DNA Transfer into Mammalian
An interesting and promising approach with respect to
Cells for Rapid Protein Synthesis
targeting is the gene-activation method. Here, an endoge-
nous, normally inactive gene (for example, the human The expression of recombinant proteins in mammalian
erythropoeitin (EPO) gene in a human tumor cell line) cells from DNA sequences that have been inserted into a
is activated by the targeted integration of a strong (viral or chromosomal site is termed stable expression. The gener-
nonviral) promoter in front of the coding sequence of the ation of stable cell lines that produce at a satisfying level
EPO gene. is usually a very time-consuming and labor-intensive pro-
cess; however, it has the advantage of providing an un-
limited basis—in time and scale—for product synthesis.
3. Amplification of Transferred DNA An alternative, more rapid, and less labor-consuming ap-
Through Methotrexate Selection
proach for protein synthesis is based on transient gene ex-
An advantage of the DHFR/CHO system for achieving pression. The concept of transient gene expression from
high productivity from chromosomally inserted DNA is mammalian cells has its molecular foundation in an effi-
the possibility of exposing recombinant cells to the DHFR cient DNA transfer to the majority of cells in a culture,
inhibitormethotrexate(MTX).Onecanelevateinstepsthe usually maintained and expanded as an adherent format
concentration of MTX in the culture medium and isolate in standard tissue culture flasks. All cells that have re-
and subsequently establish cells that have the capacity to ceived a sufficient quantity of DNA will then begin to