Mutant myeloma cells

Most current protocols use the approach of fusion with mutant myeloma cells according to a strategy in which the mutant cells are rescued from death by hybridization with lymphocytes in a medium otherwise lethal for this mutant (Littlefield, 1964 Ephrussi and Weiss, 1969) in which only the hybrids survive. Usually only a few hybrids produce the desired antibodies. 

Fig. 5.1. Two pathways in normal cells to synthesize DNA precursors. The mutant myeloma cell line, which has the capacity to grow indeflnitely in vitro and can confer this property on antibody-producing lymphocytes through cell fusion, lacks the salvage pathway (no thymidine kinase (TK) or hypoxanthine-guanine phosphori-bosyl transferase (HGPRT)). This cell line would, therefore, not grow in the presence of folic acid antagonists. Fused cells, however, may grow since the antibody-producing cells contribute the salvage pathway.

Once the fusion has taken place, it is necessary to eliminate any unfused myeloma cells and to select only hybrid cells secreting antibody. This is primarily achieved by the use of hypoxanthine aminopterin thymidine (HAT) media and cells that are deficient in the enzyme responsible for incorporation of hypoxanthine into DNA. Figure 3 illustrates this process. The unfused splenocytes are not immortal and naturally die off in culture. The elimination of the unfused myeloma cells is carried out by the initial use of mutant myeloma cells selected for a deficiency in the enzymes hypoxanthine guanine phosphoribosyl transferase (HGPRT) and thymidine kinase (TK), rendering them unable to use the salvage pathway for nucleic acid synthesis. The myelomas will die off... 

The two most popular myeloma cell lines used for this purpose are those lacking hypoxanthine-guanine phosphoribosyl transferase (HGPRT) or thymidine kinase (TK). Other kinds of mutants can be used as well, such as those resistant to ouabain (in combination with HGPRT- Baker et al., 1974). Myeloma cells treated with biochemical inhibitors can also be rescued by fusion with untreated lymphocytes (Wright, 1978). 

Mutant mouse myeloma cells unable to grow in HAT medium... 

Among the technical problems is the selection of the hybrid cells from the myeloma cells, both of which are equally rapid in growth. The biochemical trick, upon which the technique relies, is the use of mutant non-secreting myeloma cells which are deficient in hypoxanthine phosphoribosyltransferase (HPRTase ) and a culture medium containing a mixture of hypoxanthine, aminopterin and thymidine (HAT medium). Aminopterin blocks the synthesis of both purines and pyrimidines. In the presence of aminopterin, HPRTase" cells die because they cannot utilize the pertinent salvage pathway. Since B-lymphocytes contain HPRTase, the fused cells survive by utilizing the hypoxanthine and thymidine in the culture medium. The unfused B-lymphocytes are also unaffected but are rapidly outgrown by the hybrid cells. 

In the mixed population of cells resulting from fusion, the newly formed hybridomas will be HGPRT" " by inheritance from normal lymphocytes whereas the unfused myelomas carry the mutant HGPRT" marker. Therefore incubation in HAT will allow survival of the HGPRT+ hybridomas but not the HGPRT" myelomas. So, after a few days incubation in HAT the culture will contain only hybridomas. 

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