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CELL-CELL HYBRIDIZATION

Cell cell hybridization or somatic cell hybridization

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Page 1: Cell cell hybridization or somatic cell hybridization

CELL-CELL

HYBRIDIZATION

Page 2: Cell cell hybridization or somatic cell hybridization

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

Page 3: Cell cell hybridization or somatic cell hybridization

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

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Page 5: Cell cell hybridization or somatic cell hybridization

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.

Page 6: Cell cell hybridization or somatic cell hybridization

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

Page 7: Cell cell hybridization or somatic cell hybridization

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

Page 8: Cell cell hybridization or somatic cell hybridization

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

Page 9: Cell cell hybridization or somatic cell hybridization

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

Page 10: Cell cell hybridization or somatic cell hybridization

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.

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MAPPING OF GENES BY SOMATIC

CELL HYBRIDIZATION

CELL FUSION

SELECTION OF HYBRIDS

CHROMOSOME LOSSCHROMOSOMAL MAPPING

Page 12: Cell cell hybridization or somatic cell hybridization

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

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SENDAI VIRUS MEDIATED CELL FUSION

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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

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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.

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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.

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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

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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

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•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.

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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

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Page 25: Cell cell hybridization or somatic cell hybridization

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 .

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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

Page 28: Cell cell hybridization or somatic cell hybridization

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.

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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.

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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.

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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.

Page 33: Cell cell hybridization or somatic cell hybridization

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.

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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.

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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

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