Cell cell hybridization or somatic cell hybridization

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

4. 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.

5. 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

6. 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

7. 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

8. 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

9. 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.

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

11. 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

12. SENDAI VIRUS MEDIATED CELL FUSION

13. 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

14. 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.

15. 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.

16. 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

17. 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

18. 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 .

19. 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

20. 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 .

21. 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

22. 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.

23. 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 .

24. 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.

25. 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.

26. 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.

27. 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.

28. 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