Hypoxanthine aminopterin-thymidine

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... 

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... 

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. 

SV40, simian virus 40 HAT, hypoxanthine aminopterin thymidine PM A, phorbol myristate acetate LPS, lipopolysaccharide. 

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.)...

Hypoxanthine, Aminopterin, and Thymidine supplement (100X HAT, Sigma). 

Fig. 4. Multistep methods for eliciting specific antibody-producing cells (hybridomas). HAT, hypoxanthine, aminopterin, and thymidine PEG, polyethylene glycol.

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.

---