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Hybridomas, Genetic Engineering of 431

V. IMMUNIZATION IN VIVO VII. THE DEVELOPMENT
OF CELL HYBRIDIZATION
The first stage of the production of a hybridoma is to ob-
tain lymphocytes from an animal that are enriched with In 1965 Harris and Watkins reported that inactivated
specific antibody secreting cells. Immunization involves Sendai virus caused the hybridization of a mixed popu-
the injection of a chosen antigen into an animal—mice and lation of human HeLa cells and mouse Ehrlich ascites tu-
rats have been most commonly used. The time required to mor cells. The result of the fusion was a mixed population
produce an immune response resulting in antibody syn- of hybrid cells (called heterokaryons) that were geneti-
thesis will depend upon the antigen but a period of up to cally unstable. Figure 7 indicates the sequence of events
3 to 4 weeks ensures maximum response. Large molecules during fusion showing the cytoplasmic fusion of two dis-
tend to produce a strong response over a short period of similar cells followed by the hybridization of the nuclei of
time. Small molecules are often conjugated to carrier pro- the two cells. After a period of growth the heterokaryons
teins such as albumin and multiple injections spaced over tended to lose some of their genetic material and become
several days may be necessary to enhance the immune stable hybrids retaining some of the phenotypic character-
response. istics of each parental cell. The method turned out to be an
Antibodies are synthesized by B-lymphocytes which extremely valuable tool for biological research. In 1969
can be isolated from the spleen of an immunized ani- Harris showed that when normal cells were fused with
mal. The isolated spleen is homogenized gently and the malignant cells the malignant phenotype was not always
lymphocyte cell fraction collected by centrifugation. Ap- retained.Thiswasthefirstdirectevidencefortheexistence
proximately 1% of the cell population isolated from the of human suppressor genes, derived from the normal cells
spleen will secrete antibodies. At this stage the cell frac- and that could result in suppression of the tumorigenic
tion is a mixed population with a limited capacity for characteristics. These genes whose products include the
growth. retinoblastoma protein and p53 are now well characterized
in terms of their role in malignancies.
The cell hybridization technique has also been useful
VI. IMMUNIZATION IN VITRO in developing an understanding of cell differentiation and
gene regulation. For example, the normally quiescent ge-
Although most laboratories use mice for producing active netic material of highly differentiated cells can be reacti-
lymphocytes, an alternative approach involves immuniza- vated following fusion with cells actively engaged in pro-
tion in vitro. This process involves the activation of cells tein synthesis. This was shown by Harris in the late 1960s
obtained from the spleen of a non-immunized mouse. The by the fusion of a cell population of chicken erythrocytes
cells should be suspended in a medium containing the se- and growing HeLa cells.
lected antigen along with various stimulating growth and Cell fusion has also been used extensively in human
differentiation factors. These factors can be supplied from chromosome mapping. The heterokaryons resulting from
culture medium following incubation with mixed lym- the fusion of human and mouse cells are genetically un-
phocytes (or thymocytes). This is called “conditioned” stable and tend to lose human chromosomes randomly.
medium and contains various growth-promoting factors This eventually gives rise to a mixed population of sta-
secreted by the cells. These factors are called cytokines ble hybrids from which individual cell clones can be iso-
and include interleukins, B-cell growth factor and B-cell lated. Many of these clones may contain single human
differentiation factor. Some of these cytokines have now chromosomes. It is the association of a particular chromo-
been well defined and are available as recombinant pro- some in an isolated cell clone with a selected measurable
teins from commercial suppliers. The effectiveness of im- phenotypic characteristic such as an enzyme activity that
munization in vitro is dependent upon the optimal combi-
nation of these factors during cell activation.
An advantage of immunization in vitro is that the ac-
tivation of B-lymphocytes takes 3 to 4 days rather than
a few weeks as is the case with immunization in vivo.
Furthermore, weak antigens at low concentrations can
be used. A disadvantage is that certain immunoglob-
ulin isotypes tend to be produced preferentially (usu-
ally IgM) although refinement of the techniques dur-
ing cell activation can stimulate the production of other
isotypes. FIGURE 7 Cell hybridization.