Somatic cell fusion or Hybrid cells can be produced by fusing different types of somatic cells from two different tissues or species in a cell culture media. Chromosome or gene mapping via somatic cell hybridization technique is based on fusion of human somatic cell and mouse somatic cell. Usually human somatic cells like fibrocyte or leucocyte are fused with continuous cell lines of mouse.
When somatic cells of human and mouse origin or cells of any two species of mammals or two cells of same species are mixed, spontaneously cell fusion occurs at very low rate. Therefore by adding ultraviolet inactivated Sendai (parainfluenza) virus or chemical compound known as polyethylene glycol (PEG) increases the somatic cell fusion by 100 to 1000 times.
Polyethylene glycol or Sendai virus adhere to the cell membrane and alter their properties in such a way that, it facilitates cell fusion. Fusion of two different cells produces a special type of cells known as heterokaryon. That is a single hybrid cell will have two nuclei, one from each of the fused cells. After some time, the two different nuclei fuse to form a single nucleus.
Procedure for Somatic Cell Hybridization:
1. Required type of human and mouse somatic cells are selected and then mixed together along with inactivated Sendai virus or PEG (polyethylene glycol); hence they enhance the cell fusion process.
2. After some time, mixture of all three types of cell such as human cell, mouse cell and hybrid cells are plated on a selective media. Commonly used selective media is known as HAT medium. This selective media only supports the multiplication or cell proliferation of hybrid cells.
3. Some clones are extracted from the selective media and are tested for both cytogenetic and appropriate biochemical analysis for the detection of investigating biological molecule, such as enzyme or protein or any traits.
4. Now the result is correlated with the presence or absence of a trait with the presence or absence of human chromosome or gene in the somatic hybrid cells.
5. Depending upon the correlation between the presence and absence of a human chromosome or gene with that of a trait in somatic hybrid cells, can be used in locating the gene present in the concerned chromosome or genetic material.
1. The HAT medium is most commonly used selective media. This selective media is used for the selection of hybrid cells. This selective medium contains hypoxanthine, aminopterin and thymidine, hence the name. Aminopterin stops the biosynthesis of purines and pyrimidines using simple sugars and also amino acids.
2. However normal human and mouse cells still survive and replicate using hypoxanthine and thymidine which are present in the medium via salvage pathway.
3. Hypoxanthine present in the medium is converted into guanine by the help of an enzyme known as enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT), phosphorylation of thymidine is done by an enzyme known as thymidine kinase (TK).
4. Only cells with active hypoxanthine guanine phosphoribosyltransferase and thymidine kinase enzymes can survive and proliferate on a HAT medium. The cells with no active form of these enzymes will not divide in the HAT medium.
5. To use HAT medium as selective media, used human somatic cells must be deficient for any one of the two enzymes. While selected mouse cell must be deficient for the other enzyme. For example hypoxanthine guanine phosphoribosyltransferase deficient human somatic cells are fused with thymidine kinase deficient mouse cell.
6. Fusion of these somatic cells will produce hybrid cells with both the enzymes such as thymidine kinase and hypoxanthine guanine phosphoribosyltransferase and hybrid cells will proliferate on the HAT medium, whereas the human somatic cell and mouse somatic cell will not proliferate.
Uses of Hybrid Cells:
These are some of the applications of hybrid cells:
1. Hybrid cells are used to study the gene expression.
2. Hybrid cells are used to study the basics of cell division
3. Hybrid cells can be used to study the transformation of normal cells into malignant cells.
4. Hybrid cells are used to obtain viral replication
5. Hybrid cells are used for chromosome or gene mapping
6. Hybrid cells are also used in the production of monoclonal antibodies
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