Page 150 - 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 35

Hybrid cell lines carry, due to their particular type of that complex DNA molecules into a compacted structure,
creation, the chromosome sets of the two partner cells that usually together with agents that compensate its negative
were at the origin of the fusion. One of the two partners charge. The most popular of the complexing agents is cal-
is a cell of immortal character (usually a tumor-derived cium phosphate, which can be precipitated in the presence
cell line), and these cells are generally known to exhibit of DNA and generates microscopic particles (up to 3 µm
a high degree of cytogenetic instability, a feature that is in size, but preferably smaller than 0.3 µm at the time of
transferred into the fusion product. A disadvantage of hy- interaction with the cells). Small calcium phosphate DNA
bridoma cell lines, in comparison with, for example, trans- co-precipitates(smallerindiameterthan0.3 µm)cancarry
fected Chinese hamster ovary (CHO) cell lines expressing as much as 40% of their entire mass as DNA and individ-
an antibody, is an inherent (productivity) instability. This ual 0.5-µm particles can carry as many as 4500 copies of a
instability is due to the decline and eventual overall loss standard-sized plasmid DNA. More recently, other lipid-
of cells in the cell population expressing the desired an- or polymer-based DNA delivery vehicles have been devel-
tibody and an overgrowth of nonproducing clones in the oped, most of them provided now in the form of commer-
cell population. In order to compensate for this disad- cial kits. As with calcium phosphate DNA co-precipitates,
vantage, single-cell cloning and expansion from “young” the majority of these polymer- or lipid-based vehicles de-
hybridomas are frequently necessary while limiting the liver DNA into endosome compartments of the cells. It
subcultivation of cell populations to short time frames. To is assumed that the transfer across the cellular membrane
obtain a reasonable number of cell lines expressing the de- occurs by endocytosis. The different steps of transport and
sired antibody, several cloning steps with candidate fusion processing of DNA to finally achieve expression from the
product cells have to be executed. DNA sequences integrated into the genome (see Fig. 1) are
Monoclonal antibodies derived from murine hybrido- poorly understood. They are a subject of intense research
mas have been used in a variety of clinical applications, due to increased interest in gene therapy, in which genes
but some of the early promise could not be realized due to need to be delivered to the nucleus of many individual
human anti-mouse immune reactions induced in treated cells of the patient.
patients. The concept of fusion of two biological systems
for the synthesis of a specific monoclonal antibody has
2. Transfer of Genes of Interest
also been used for the creation of murine–human het-
into Mammalian Cells
erohybridomas and for human–human hybridomas. In all
these cases, because of the use of cells derived from blood The generation of recombinant cell lines (i.e., cell pop-
sources, a very important characteristic has been “inher- ulations that have incorporated into one or more of their
ited” that significantly facilitates the scale-up of the cell chromosomes segments of DNA provided experimentally
substrate to very large cell number—the growth of single through viral or nonviral vectors) has been motivated by
cells in free suspension. the desire to understand phenomena of gene expression,
gene control, and gene regulation. During the early 1980s,
mammalian cells and, in particular, CHO cells with a
2. Heterokayons double mutation/deletion of the dihydrofolate reductase
Somatic cell fusion using Sendai virus or polyethylene (DHFR) gene locus became a rather convenient substrate
glycol, and a selective medium to prevent the growth of the for the introduction of genes. The availability of these
parent (non-hybrid) cells, was a valuable tool for studying mutant CHO cells allowed identification, in selective me-
somatic cell genetics in the 1960s and 1970s. The devel- dia lacking certain precursors for nucleotide synthesis, of
opment of hybridoma technology used these techniques, clones of cells that express the DHFR gene. The DHFR
but currently recombinant genetic techniques have largely gene can be provided by transfection with a plasmid vec-
replaced the classical fusion techniques. However, cell fu- tor into which another gene of interest has been cloned
sion is still used to study fundamental genetics and physi- or to which a second plasmid containing that gene of in-
ology, as well as the more publicized monoclonal antibody terest was added for the preparation of the transfection
technology. cocktail (Fig. 2). In both cases, emerging cells after se-
lection contain the DHFR gene and the gene of interest
in their chromosomes, usually genetically closely linked
D. Recombinant Cell Lines to each other. The purpose of most of these experiments
has been and still is to obtain cell lines that produce the
1. Nonviral DNA Transfer Vehicles
product of the gene of interest at high levels in a stable
for Mammalian Cells
manner. In general, CHO cells are considered cytogeneti-
The transfer of genes of interest into mammalian cells cally stable, a feature that translates to recombinant CHO
was greatly facilitated by the development of methods cells.