Cell hybrids fusion

To obtain MABs, lymphocytes isolated from the spleen of immunized mice (1) are fused with mouse tumor cells (myeloma cells, 2). This is necessary because antibody-secreting lymphocytes in culture have a lifespan of only a few weeks. Fusion of lymphocytes with tumor cells gives rise to cell hybrids, known as hybridomas, which are potentially immortal. 

Figure 16.1. Schematic representation of the yeast two-hybrid system for evaluation of protein-protein interactions. Haploid yeast of a and a cells can mate to form (a/a) diploid cells. (A) If two test proteins, PT1 and PT2—expressed in (a/a) diploid cells as fusion proteins of DNA binding domains (DAB) and activation domains (AD) of yeast gene-transcript activator proteins—bind to each other, the binding interaction allows the diploid cells to grow in histidine selection media. Histidine selection media is permissive for diploid cells that express the HISS reporter gene only if PT1 and PT2 interact. (B) If PT1 and PT2 do not interact, no HISS gene product is expressed and the hybrid cell cannot grow in histidine media.

Cell Fusion Unlike antibody-secreting cells, myeloma cells, malignant tumor cells of the immune system, can be cultured continuously. Kohler and Milstein (1975) developed a method to fuse (hybridize) B-lymphocytes from the mouse spleen with mouse myeloma cells, so that the fused cell, hybrid-myeloma (or hybridoma) cell, can have the characteristic of the both cell lines that is, the production of specific antibodies and the immortality. Since the hybridoma is derived from a single B-lymphocyte, it produces only one kind of antibody, thus a monoclonal antibody. 

Cultivate a suitable malignant myeloma cells deficient in HPGRT (hypoxanthine guanine phosphoribosyl transferase), which is a genetic marker for the selection of the hybrid cells after fusion. 

Integral proteins are usually free to move in the plane of the bilayer by lateral and rotational movement, but are not able to flip from one side of the membrane to the other (transverse movement). Immunofluorescence microscopy may be used to follow the movement of two proteins from different cells following fusion of the cells to form a hybrid heterokaryon. Immediately after fusion the two integral proteins are found segregated at either end of the heterokaryon but with time diffuse to all areas of the cell surface. The distribution of integral proteins within the membrane can be studied by electron microscopy using the freeze-fracture technique in which membranes are fractured along the interface between the inner and outer leaflets. 

Cell hybridization The fusion of two or more dissimilar cells leading to the formation of a synkaryon. 

Somatic cell hybrid The cell resulting from the fusion of animal cells derived from somatic cells that differ genetically. 

The Biological Station of the Institute for Immunology of the Eotvos Lorand University (ELTE) in God started an intensive study on cell-hybridization and cell-fusion in higher-order mammals in order to promote diagnostic investigations. As a result, antibodies prepared by the team were soon figuring in WHO lists. 

In summary, although the application of Y3H may be limited in some scenarios, most of these are likely to be rare events. The most limiting factor is likely the requirement for expression of fusion proteins that are able to translocate into the nucleus of yeast cells while retaining a properly folded small molecule binding domain. This may, however, not be an issue with many proteins, because of their modular structure. A modular structure favors proper folding of a binding domain, even when it is expressed in isolation or as part of a hybrid fusion protein. Thus, the use of complex cDNA libraries, which contain multiple fusion variants of a particular protein, is preferable and will decrease the occurrence of false negatives. 

Tarshis M, Salman M, Rottem S. Fusion of mycoplasmas the formation of cell hybrids. FEMS Microbiol Lett. 1991 66 67-71. 

This granted, one of the simplest (and probably least enlightening) possibilities is that the cause of some or all the anomalies described (and particularly those expressed as the directly observed mitotic asynchrony) resides in the origin of hybrid cells from fusion of pairs of cells in very different phases of the cell cycle. 

Mammalian Cells Unlike microbial cells, mammalian cells do not continue to reproduce forever. Cancerous cells have lost this natural timing that leads to death after a few dozen generations and continue to multiply indefinitely. Hybridoma cells from the fusion of two mammalian lymphoid cells, one cancerous and the other normal, are important for mammalian cell culture. They produce monoclonal antibodies for research, for affinity methods for biological separations, and for analyses used in the diagnosis and treatment of some diseases. However, the frequency of fusion is low. If the unfused cells are not killed, the myelomas 1 overgrow the hybrid cells. The myelomas can be isolated when there is a defect in their production of enzymes involved in nucleotide synthesis. Mammahan cells can produce the necessary enzymes and thus so can the fused cells. When the cells are placed in a medium in which the enzymes are necessaiy for survival, the myelomas will not survive. The unfused normal cells will die because of their limited life span. Thus, after a period of time, the hybridomas will be the only cells left ahve. 

The use of protoplasts in studies of stress physiology and biochemistry expands the advantages of cell culture systems discussed in the preceding sections. Additional applications are related to the fusion of protoplasts. Intraspecifie and interspecific protoplast fusion greatly enhance genetic variability of the fused protoplasts (Kumar Cocking, 1987). The resulting somatic hybrids provide cells which can be used for selection of specific traits (e.g. environmental stress tolerance) provided by one or both donor cells and for basic studies on cytoplasmic and nuclear inheritance of desired characteristics. 

---