HAT MEDIUM SELECTION:

HAT selection depends on the fact that mammalian cells can synthesize nucleotides by two different pathways: the de novo and the salvage pathways. The de novo pathway in which a methyl or formyl group is transferred from an activated from of tetrahydrofolate, is blocked by Aminopterin, a folic acid analog. When the de novo pathway is blocked, cells utilize the salvage pathway, which bypasses the aminopterin block by converting purines and pyrimidines directly into DNA. The enzymes catalyzing the salvage pathway include hypoxanthine-guanine phosphorribosyl transferase (HGPRT) and thymidine kinase (TK). A mutation in either of these two enzymes blocks the salvage pathway. HAT medium contains Aminopterin to block the de novo pathway and hypoxanthine and thymidine to allow growth via the salvage pathway. When two types of cells, one with a mutation in TK and the other with a mutation in HGPRT are fused, only the hybrid cells will contain the full complement of necessary enzymes for growth on HAT medium via the salvage pathway. Thus only hybrid cells will grow in HAT medium.