Hypoxanthine aminopterin thymidine medium

Conversely, HPRT4 cells can be selected for in the presence of hypoxanthine-aminopterin-thymidine medium. It should be possible to select for HPRT cells in normal HPRT+ persons with one of the analogues, and this was one of the first systems used to detect spontaneously occurring mutants in freshly cultured cells from humans.88 388... 

In order to determine whether restriction is due to a lack of host factor as opposed to inhibition by a host factor of viral functions, it was decided to hybridize MDBK with L-cells. We have introduced an HGPRT mutation into the MDBK cells, and utilized a TK strain of L-cells. Hybrids were selected in HAT medium (hypoxanthine, aminopterin, thymidine), clones were selected, grown up, and tested for virus yield. Table 2 summarizes the data obtained. 

The cell fusion mixture is transferred to a culture medium containing hypoxanthine, aminopterin and thymidine (HAT medium). Unflised myeloma cells are unable to grow as they lack HGPRT. Unflised normal spleen cells can grow but their proliferahon is limited and they eventually die out. The hybridoma cell can proliferate in the HAT medium as the normal spleen cell supplies the enzyme which enables the hybridoma to utilize extracellular hypoxanthine. 

Successful fusion (2) is a rare event, but the frequency can be improved by adding polyethylene glycol (PEG). To obtain only successfully fused cells, incubation is required for an extended period in a primary culture with HAT medium (3), which contains hypoxan-thine, aminopterin, and thymidine. Amino-pterin, an analogue of dihydrofolic acid, competitively inhibits dihydrofolate reductase and thus inhibits the synthesis of dTMP (see p. 402). As dTMP is essential for DNA synthesis, myeloma cells cannot survive in the presence of aminopterin. Although spleen cells are able to circumvent the inhibitory effect of aminopterin by using hypoxanthine and thymidine, they have a limited lifespan and die. Only hybridomas survive culture in HAT medium, because they possess both the immortality of the myeloma cells and the spleen cells metabolic side pathway. 

Fusion with the cells compensates for this deficiency. When fused and unfused cells are incubated in the presence of the folic acid antagonist aminopterin, the de novo synthesis of purines and pyrimidines for DNA is blocked. Cells deficient in HGPRT die, whereas hybrid cells are able to bypass aminopterin blockage by metabolism of hypoxanthine and thymidine added to the medium. In the generation of mouse hybridomas, an number of myelomas deficient in HGPRT are available, all originating from MOPC 21, a spontaneous myeloma from the BALB/c mouse strain. 

Select HPGRT+ cells by culturmg the mixture in a medium containing HAT (hypoxanthine, aminopterin, and thymidine), which is growth inhibitory to HPGRT+ cells. Therefore, the myeloma cells will die in this medium while the hybridoma cells proliferate. The unfused lymphocytes will die due to their limited lifespan. 

HGPRT and TK enzymes are important in the synthesis of DNA from preformed nucleotides provided in the culture medium. The myeloma cells lacking these enzymes are unable to utilize the hypoxanthine or thymidine from the medium moreover, they die in the presence of aminopterin, which inhibits the de novo synthesis of DNA. Thus, in a medium containing aminopterin, hypoxanthine, and thymidine (HAT medium) only those hybridomas that receive the HGPRT or TK enzyme from the normal parents (spleen B lymphocytes) will survive. 

Fig. 13.3. Metabolic cooperation between BHK21/C3 cells. Growth of BHK-HPRT-and BHK-TK- cells separately and in mixed (1 1) culture, in medium containing hypoxanthine, aminopterin and thymidine (HAT medium). HPRT = hypoxanthine phosphoribosyl transferase TK = thymidine kinase. (Reproduced from Pitts, 1971, with kind permission of the author and publisher.)...

Hybridomas are generated 3 d following an iv booster injection of 10 pg of DT in phosphate-buffered saline (PBS) and selected using Hy medium with 10% fetal calf serum plus hypoxanthine, aminopterin, and thymidine (HAT) (6,16). 

HAT Medium - A cell culture medium augmented by hypoxanthine, aminopterin, and thymidine, which selects for cells that have a functional purine salvage pathway. 

HAT Selection - The compounds hypoxanthine, aminopterin (see here), and thymidine (H,A, and T, respectively) can be used to select for cells having functional salvage pathways. Aminopterin inhibits dihydrofolate reductase, which blocks de novo purine and thymidine synthesis. Only cells which can utilize thymidine (pyrimidine salvage) and hypoxanthine (purine salvage) can grow in this medium. 

FIGURE 52-2 Generation of monoclonal antibodies. Mice are immunized with the selected antigen, and spleen or lymph node is harvested and B cells separated. These B cells are fused to a suitable B-cell myeloma that has been selected for its inability to grow in medium supplemented with hypoxanthine, aminopterin, and thymidine (HAT). Only myelomas that fuse with B cells can survive in HAT-supplemented medium. The hybridomas expand in culture. Those of interest based upon a specific screening technique are then selected and cloned by limiting dilution. Monoclonal antibodies can be used directly as supernatants or ascites fluid experimentally but are purified for clinical use. HPRT, hypoxanthine-guanine phosphoribosyl transferase. 

Figure 3 Schematic representation of HAT selection. HAT medium contains aminopterin to block the de novo pathway and hypoxanthine and thymidine to allow growth by the salvage pathway. Cells that lack either HGPRT or TK will die in HAT medium, because they lack the ability to use the salvage pathway to synthesize nucleic acids.

The principle of selection is to place these cells into a selective medium in which the de novo pathway of nucleotide synthesis is inhibited. The key to this is the compound, aminopterin which is an analogue of folic acid and a specific inhibitor of dihydrofolate reductase, an essential enzyme for the formation of tetrahydrofolate (FH4) required as a coenzyme of the de novo purine nucleotide synthesis pathway. Tetrahydrofolate is also required for the formation of thymidine. However, if hypoxanthine and thymidine are provided in the culture media of HGPRT+ cells they will be able to grow normally. On the other hand HGPRT" cells would have no means of synthesizing purine nucleotides and consequently would be unable to grow. 

The selective medium, HAT contains hypoxanthine, aminopterin, and thymidine. This medium allows the selection and growth of hybridomas which are HGPRT+ and have a normal functioning salvage pathway. However, HAT is unable to support the growth of the HGPRT" myelomas because the de novo pathway is inhibited and the salvage pathway cannot function because of the defective enzyme. 

Concentrations of deoxycytidine, hypoxanthine, and thymidine that sustain normal growth of MIT-2, WI-L2, and TK6 lymphoblasts in the presence of 2 X 10 M aminopterin are 10 M, 2 X 10 M, and 1.75 X 10 M, respectively. These concentrations were empirically determined. We refer to this mixture as CHAT and the corresponding medium without aminopterin as THC. 

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. 

HAT (HT) medium Medium containing hypoxanthine, aminopte-rin, and thymidine (HT without aminopterin)... 

F-11 cells were cultured in DMEM/F12 medium +20% FBS, 0.2 mM L-glu-tamine, 100 pM sodium hypoxanthine, 400 nM aminopterin, 16 pM thymidine (HAT supplement), and penicillin/streptomycin at 37°C (the cells were kindly provided hy Dr. S.E. Gordon, University of Washington). 

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