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432 Hybridomas, Genetic Engineering of

allows the gene of that enzyme to be assigned to a spe- Alternatively, electrofusion can be used. In this tech-
cific human chromosome. With the use of this technique, nique, two populations of cells are introduced into a small
many human genes have been assigned to particular chro- sterile chamber. An electric current is applied in high-
mosomes (known as “chromosome mapping”). voltage pulses for short time periods. During this period
It was this same technique of cell fusion that Kohler and the membrane will become highly permeable. This is sim-
Milstein used in their work reported in 1975 that allowed ilar to the process of electroporation used to facilitate the
the creation of stable hybrid cells from the hybridization entry of DNA into cells. This causes the cells to orientate
of antibody-secreting B-lymphocytes with transformed alongthelineofthecurrentandfuse.Thisprocessishighly
myelomas. The resulting cells retained two important phe- efficient, producing a high percentage of viable hybrid
notypic characteristics from the parents—the ability for cells. The most suitable voltage for electrofusion is one
infinite growth (from the myeloma) and the ability to syn- that causes approximately 50% death in the cell popula-
thesize antibody (from the lymphocyte). The original ob- tion. This would typically be around a voltage of 200 V for
jective of this work was to study somatic mutation as a a cell pellet held in a small electroporation cuvette. From
mechanism for antibody diversity. This is the ability of such a protocol there may be around 50 fused cells from an
∧
B-lymphocyte to go through a maturation process follow- original total of 5 × 10 6 cells from each parental cell line.
ing initial contact with an antigen to produce antibod-
ies of increasing affinity. However, the application of the
cell fusion technique to produce antibody-producing cells IX. CELL FUSION TO IMMORTALIZE
with an infinite growth capacity had a major impact on LYMPHOCYTES
the ability to produce large quantities of antibodies that
could be used for a variety of functions both in biologi-
Although immunization can result in lymphocytes capa-
cal research and also as medically important products. The
ble of producing the required antibody, the cells will only
term “hybridoma” was derived in 1976 by Herzenberg and
grow for a limited period of time. The purpose of lympho-
Milstein to describe a homogeneous clone of these
cyte hybridization is to combine the desired property of
antibody-producing hybrid cells. The term “monoclonal
antibody synthesis of the B-lymphocyte population with
antibody” refers to the secreted product of the cells. Unlike
the infinite growth capacity of a myeloma. Therefore,
antibodies derived from blood samples (“polyclonal”),
the selected lymphocytes are fused with a population of
the monoclonal antibodies from a single hybridoma are
myeloma cells. Those commonly used for mouse or rat
molecularly homogeneous and have a specificaffinity for
cells are shown in Table I. Suitable myeloma fusion part-
a particular antigen.
ners are selected for two other important characteristics:
Nonproduction of antibodies. This is desirable so that
VIII. METHODS OF CELL FUSION
the resulting hybridoma does not synthesize more than
one antibody.
Cells can be induced to fuse if two cell populations are
Possession of a genetic marker, such as the lack of an
∧
brought close together at a high cell concentration (10 6
enzyme, to allow cell selection. For example,
to 10 7 cells per well of a 96 multi-well plate) in the
∧
myelomas deficient in HGPRT (hypoxanthine guanine
presence of viruses or by chemical agents (called “fuso-
phosphoribosyl transferase) are commonly used. This
gens”). The process involves a destabilization of adjacent
allows selection of hybridomas in HAT medium (see
cell membranes which eventually fuse to form a hybrid
“selectable gene markers”).
cell. Initially, two distinct nuclei are present in the fused
cell (a heterokaryon). Eventually the nuclei fuse to pro-
duce a stable hybrid cell.
Although UV-inactivated Sendai viruses were origi- TABLE I Rodent Cell Lines (Myelomas)
Commonly Used as Fusion Partners
nally used as agents for cell fusion, the more widely used
method is now fusion by the chemical agent polyethylene Immunoglobulin
glycol (PEG). This is a polymer, available at a molecular Species Cell line expression
weight range of 200–20,000 kD. PEG at 4000–6000 kD
Mouse X653 No
is most suitable for cell fusion. Cell fusion can occur in a
NS0 No
solution of PEG (40–50% w/v) within 1–2 min. In this pro-
Sp2/0 No
cess, cell swelling accompanies fusion. This enables ad-
NS1 Yes
jacent cells to approach very closely and also the plasma
Rat YB2/0 No
membrane becomes permeable to small ions. However,
Y3-Ag Yes
lysis of swollen cells may also occur.