Hybridoma Selection

Cell division requires an adequate supply of free nucleotides for DNA replication. Nucleic acids are generated either via:

  • De novo DNA synthesis – which is dependent on the activity of dihydrofolate reductase to generate purine nucleotides (GMP, IMP, AMP) and thymidylate;
  • Salvage pathway synthesis – which requires exogenous hypoxanthine and thymidine and the enzymes hypoxanthine-guanine phosphoibosyltransferase (HGPRT) and thymidine kinase (TK).

Aminopterin is a dihydrofolate reductase inhibitor: treatment of cells with aminopterin prevents de novo DNA synthesis, and in the absence of exogenous hypoxanthine and thymidine to supply the salvage pathway, the cells will die. A mutation in HGPRT disables the salvage pathway, which is lethal in aminopterin-treated cells even if they are cultured with exogenous hypoxanthine and thymidine. The identification of myleoma cells with mutations in HGPRT is straightforward. The HGPRT gene is on the X-chromosome, and due to X-linked inactivation a single mutation is all that is needed to result in the loss of HGPRT. Thus, specific cell culture conditions (HAT media: Hypoxanthine, Aminopterin, Thymidine) + a myeloma partner with a HGPRT mutation enables the selection and survival of hybridomas (B cell:myeloma hybrids) vs. unfused myeloma cells or B cells. The key concept is that the hybridoma receives a functional HGPRT gene from the primary B cell, thus enabling the survival of hybridomas vs. myeloma cells.  While primary B cells can survive for a time in culture and produce antibody, they will eventually die without any additional selection.

APRT, adenine phosphoibosyltransferase; PRPP, 5-Phosphoribosyl-1-pyrophosphate.

* Modified from Hnasko et al, “Hybridoma Technology”, Methods in Molecular Biology 2015.