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

36 Mammalian Cell Culture

FIGURE 1 Transfection barriers: DNA for integration into chromosomes of mammalian cells has to overcome various
barriers. The steps involved are numbered: (1) Neutralization and compactation of DNA, (2) association with cellular
membrane and endocytotic entry of compacted DNA, (3) endosomal transfer towards nucleus and partial degradation
(not shown) of DNA, (4) entry of DNA into nuclear environment, and (5) integration of DNA into chromosomal DNA.

Besides DHFR, drugs such as neomycine, hygromy- mosomal locus that is different for each clone. These two
cine, and puromycine can be used in combination with factors of variability in gene integration and probably a
plasmids that confer resistance to these components for number of other, so far unknown phenomena make it nec-
the selection and identification of recombinant cells. A essary to analyze many clonal cell lines to identify a few
positive selection mechanism is based on the use of the that meet the expected expression levels. With moderate
glutamine synthetase (GS) gene in transfected plasmids screening and nonoptimized vectors, expression levels of
when growing CHO cells in medium lacking glutamine. 0.1 to 10 pg of recombinant protein/cell/24 h can be con-
The GS system can be applied to other non-CHO cell sidered acceptable for secreted proteins. However, from
lines and also allows the amplification of the transferred more stringently screened clones, including those that de-
expression vectors in the genome of these cells. rive from methods that increase the copy number of inte-
The integration of exogenous DNA in the chromosomes grated DNA molecules (see below), expression levels of
of mammalian cells is poorly understood and, when using 10 to 50 pg/cell/24 h can be obtained.
standard transfection and selection procedures, is a largely Another approach to possibly improve methods for
uncontrolled process. A major reason for observed vari- identification of high-level expression from transgenic
ability in expression of clones isolated upon transfection is DNA in mammalian cells and to maintain better con-
the variation in copy number and in location of integrated trol over the outcome of the gene transfer experiments
plasmid molecules. In spite of usually large excesses of is targeting into defined chromosomal sites. Here, prefer-
plasmid molecules entering the cells upon transfection entially chromosomal areas of high transcription activity
(10,000 to 100,000 copies), variable but small numbers are desired, as are compensating or preventing trends in
of plasmid copies (1 to 100) integrate into a single chro- chromosomal silencing frequently observed when DNA