What is Cell-Cell Hybridization? History More about Somatic cell Hybridization Mapping of genes by somatic cell Hybridization Hybridoma technology Other Applications of Somatic Cell Hybridization

1. CELL-CELL
HYBRIDIZATION

2. CONTENTS
 What is Cell- Cell Hybridization?
 History
 More about Somatic cell Hybridization
 Mapping of genes by somatic cell Hybridization
 Hybridoma technology
 Other Applications of somatic Cell Hybridization
 Conclusion
 Reference

3. WHAT IS CELL- CELL
HYBRIDIZATION?
– CELL CELL HYBRIDIZATION:
– The technique of hybrid production through the fusion of somatic
cells under in vitro conditions, is called Somatic Hybridization
– Somatic Cell Hybridization involves the fusion of two different cells
to produce a hybrid which is termed as Heterokaryon
– The product of fusion was called Homokaryon if the two parental
cells come from the same species

5. Creation of a PC-12 homokaryon. The cells were originally located at a distance of about 100 μm
from each other. By using the microelectrodes, they were placed next to each other. The cells were
pretreated with di-electrophoresis . A shows cells immediately after di-electrophoresis, and a close
contact is visualized by the flattened contact zone between the cells.

6. HISTORY
• In vitro somatic cell hybridization was first discovered by George Barski in
Paris in 1960
• Barski reported that cultures containing a mixture of two sublines of
heteroploidy mouse cell were overgrown by new cell lines, the karyotype of
which was the sum of two parental karyotypes
• Lederberg(1958) and Pontecorvo in 1961 sensed the potential of this new
approach.
Ephrussi confirmed the validity of the observation by Barski’s Group

7. MORE ABOUT SOMATIC
HYBRIDIZATION
-Somatic cell hybridization are of two types
• Spontaneous Hybridization is very rare
• -Induced Hybridization is mediated by either chemically with Polyethylene
Glycol, which effects the cell membrane, or with inactivated virus for
example the Sendai Virus.
SPONTANEOUS INDUCED

8. MORE ABOUT SOMATIC CELL
HYBRIDIZATION
• Hybridization can either be performed between two same species
which is termed as interspecific
• Or it can also be performed between two different species
Intraspecific

9. SOMATIC HYBRIDIZATION IN
ANIMAL CELL
– Somatic cells of different types can be fused to obtain hybrid
cells. Hybrid cells are useful in a variety of ways, e.g.,
– (i) for gene or chromosome mapping
– (ii) production of monoclonal antibodies by
producing hybridoma

10. GENE MAPPING BY SOMATIC
CELL HYBRIDIZATION
– The ability to distinguish each human chromosome is required to perform somatic-cell
hybridization, in which human and mouse (or hamster) cells are fused in culture to form a
hybrid.
– The fusion is usually mediated chemically with polyethylene glycol, which affects cell
membranes; or with an inactivated virus, for example the Sendai virus, that is able to fuse
to more than one cell at the same time.
– When two cells fuse, their nuclei are at first separate, forming a heterokaryon, a cell with
nuclei from different sources.

11. MAPPING OF GENES BY SOMATIC
CELL HYBRIDIZATION
CELL FUSION
SELECTION OF HYBRIDS
CHROMOSOME LOSS
CHROMOSOMAL MAPPING

12. CELL FUSION
– The commonly used fusion agents for mapping of chromosome are Sendai virus,
lysolecithin, liposomes and polyethylene glycol (PEG).
– The most widely used fusion agent is Sendai virus, which is inactivated by UV light or
β-propiolactone
– The process of fusion by Sendai virus involves 3 stages –
 The virus particles cause cell agglutination
 Cytoplasmic bridges are formed between cells
 Cytoplasmic bridges expand to form spherical fused cells

14. SENDAI VIRUS MEDIATED CELL FUSION

15. SELECTION OF HYBRIDS
– To isolate pure population of Human –Mouse Heterokaryon a selection procedure is used
that kills both the parental cells and the homokaryons but allows the human – mouse
hybrid cells to survive and grow.
– In the medium contains a drug aminopterin, which blocks the de novo purine and
pyrimidine biosynthetic pathways of cells.
– However , the presence of hypoxanthine and thymidine the cells can overcome the block
by synthesizing their purines and pyrimidines using salvage pathways

17. SELECTION OF HYBRIDS
– For cells to grow in HAT medium, two enzymes, hypoxanthine-guanine
phosphoribosyl transferase (HGPRT) and thymidine kinase (TK) must be functional.
– Mouse cell is deficient in TK (TK-) and a human cell is deficient in HGPRT(HGPRT-)
– Heterokaryons , has normal HGPRT gene derived from the mouse genome and TK
from gene from the human genome. Therefore only hybrid cell grows.

18. CHROMOSOME LOSS IN
HYBRID CELLS
– When hybrids are formed between rodent and human cells , human
chromosomes are lost preferentially.
– Loss of chromosome in interspecific hybrids was noted in fusion between
mouse and rat cells., where chromosomes of rat were preferentially lost.
– In rodent and human cell fusions, up to 95% of the human chromosomes
can be lost by the time the hybrids are initially isolated and their
chromosome analyzed.

20. MAPPING STRATEGIES
– The qualities of rodent –human hybrids that make useful for
human mapping are-
– Preferential loss of human chromosomes (in most cases)
– Easy detection or unilateral loss of human phenotypes and
differentiation from their rodent counterpart

21. SYNTENY TESTING
– This is based on the premise that genes located on the same chromosome
will be retained and lost concordantly from hybrid cells
– Gene pairs or groups were determined to be syntenic by observing the
pattern of retention and loss in a large number of independently derived
hybrid cell line.
– However this must be supported by chromosomal analysis to rule out loss of
a pair of chromosomes or extensive chromosome rearrangements that would
obscure a proper syntenic relationship

22. •Product A can be assigned to chromosome 5.
• Product B can be assigned to chromosome 3.
• Product C is not on any of the chromosomes 1-7.
• Product D can be assigned to chromosome 1.

23. ASSIGNMENT BY SELECTION
– The first assignment of genes to chromosomes using rodent-human hybrids
relied on the ability to select for the genes.
– The assignment of the TK gene to the chromosome 17 was facilitated by
isolation of reduced which contained only chromosome 17 in common.
– TK- mouse cells were fused with human cells in HAT medium and the hybrid
cells were examined, and only chromosome 17 was found to be common,
which would be carrying the TK gene

25. BASIC HYBRIDOMA
TECHNOLOGY
– Hybridomas are cells that have been engineered to produce a desired antibody in
large amounts, to produce monoclonal antibodies.
– Monoclonal antibodies can be produced in specialized cells through a technique now
popularly known as hybridoma technology.
– Hybridoma technology was discovered in 1975 by two scientists, Georges Kohler of
West Germany and Cesar Milstein of Argentina .

27. STAGES
– The method developed by Kohler and Milstein involves four stages which
results in the production of hybrid lymphocyte with an infinite growth
capacity and capable of continuous of a single antibody.
– The stages of this process are shown:
 Immunization
 Cell fusion
 Genetic Selection
 Cell selection

28. IMMUNIZATION
– The first stage of production of a hybridoma is to obtain lymphocytes
from and animal that is enriched with specific antibody- secreting
cells.
– Antibodies are synthesized by B lymphocytes which can be isolated
from the spleen of an immunized animal.

29. METHODS OF FUSION:-
– Cells can be induced to fuse if two cell populations are brought close together at
a high cell concentration in the presence of viruses or by chemical agents (called
‘fusogens’).
– The process involves the destabilization of adjacent cell membranes which
eventually fuse to form a hybrid cell.
– Although UV-inactivated Sendai viruses were originally used as agents for cell
fusion, the more widely used method is now fusion by the chemical agent
polyethylene glycol (PEG). This is a polymer, available at a molecular weight
range of 200–20000 kDa.

31. SELECTABLE GENE MARKERS
FOR CELL SELECTION
– The process of cell fusion will result in a heterogeneous population
of cells that will contain unfused parental cells, lysed cells as well
as the required hybrid cells. At this stage, cell selection is
important so that the hybrid cells can be isolated from the
mixture. For hybridomas there are two important stages of cell
selection:
■ isolation of hybrid cells from parental cells;
■ selection of antibody-secreting cells within the hybrid cell
population.

32. Clonal selection of Mab-secreting
hybridomas
• After genetic selection with HAT the culture contains hybridomas but only some of
these will secrete antibodies.
• The next stage involves selection of Mab-secreting hybridomas from the
population which has survived HAT treatment Cell clones can be isolated by the
method of limiting dilution.
• Cloning ensures that all cells selected for future cultures are genetically identical.

33. OTHER APPLICATIONS OF
SOMATIC CELL HYBRIDIZATION
– 1. Somatic hybridization has helped to study the cytoplasmic genes and their functions.
In fact, the information is successfully used in plant breeding programmes.
– 2. Protoplast fusion will help in the combination of mitochondria and chloroplasts to
result in a unique nuclear-cytoplasmic genetic combination.
– 3. Somatic hybridization can be done in plants that are still in juvenile phase.
– 4. Protoplast transformation (with traits like nitrogen fixation by incorporating
exogenous DNA) followed by somatic hybridization will yield innovative plants.

34. CONCLUSION
– Somatic Cell Hybridization is the process of fusion which takes place between
two Somatic Cell.
– Somatic cell hybridization is usually done between mouse and human cells, but
many other intraspecific or interspecific hybridizations can be done.
– It is widely used for the production of Monoclonal Antibodies and for the Gene
Mapping of Human Chromosomes.
– Even though this presentation specifies on animal cell, this process can be used
for plant cell hybridization.

35. REFERENCE
- Brackett B. J., New technologies in animal breeding, Elsevier 2012 pg- 171- 193
– Jaffe E. A., Biology of Endothelial cells, Springer Science and Business Media,2012. Pg- 194
– Lodish H, Berk A, Zipursky SL, et al., 2000. Molecular Cell Biology, 4th edition, W. H. Freeman.
– Ephrussi B., Hybridization of Somatic Cells, Princeton University Press 2015. pg-166
– Crow J. F. and Dove W. F., 2000. Perspectives on Genetics: Anecdotal, Historical, and Critical Commentaries
1987-1998, Univ of Wisconsin Press
– McConkey E H,, 1993. Human Genetics: The Molecular Revolution, Jones & Bartlett Learning
– Yunis J., 2012. Molecular Structure of Human Chromosomes, Elsevier.
– Meurant G., (1977). Methods in Cell Biology, Volume 15, Academic Press