- HAT medium
HAT Medium (
Hypoxanthine Aminopterin Thymidine medium) is a selection medium for mammalian cell culture, which relies on the combination of aminopterin, a drug that acts as afolate metabolism inhibitor by inhibitingdihydrofolate reductase , withhypoxanthine andthymidine , which are intermediates inDNA synthesis - apurine derivative and a deoxynucleoside , respectively. The trick is that aminopterin blocks DNA "de novo" synthesis, which is absolutely required forcell division to proceed, but the other components provide cells with the raw material to evade the blockage (the "salvage pathway") - provided that they have the rightenzymes , which means having functioning copies of thegenes that encode them.The enzyme
dihydrofolate reductase which producestetrahydrofolate (THF) by the reduction of dihydrofolate,is specifically blocked by aminopterin. THF, acting in association with specificprotein s, can receive single carbon units that are then transferred to specific targets.One of the important targets for cellular reproduction is
thymidylate synthase , which createsthymidine monophosphate (TMP) fromdeoxyuridine monophosphate (dUMP). By additionalphosphorylation reactions TMP can be used to makethymidine triphosphate (TTP), one of the four nucleotide precursors that are used byDNA polymerases to createDNA . Without the THF required to convert dUMP, there can be no TTP, and DNA synthesis cannot proceed --- unless TMP can be produced from another source. The alternative source is thatthymidine present in HAT medium can be absorbed by the cells and phosphorylated bythymidine kinase (TK) into TMP.The synthesis of IMP, (precursor to GMP and GTP) also requires THF, and also can be bypassed. In this case
hypoxanthine-guanine phosphoribosyltransferase (HGPRT) reacts hypoxanthine absorbed from the medium withPRPP , liberatingpyrophosphate , to produce IMP by a salvage pathway.Thus the use of HAT medium for cell culture is a form of
artificial selection for cells containing working TK and HGPRT. Many useful refinements to the scheme are made possible by poisons that kill cells, but to which they are immune if they lack one of these genes. Thus a cell lacking TK is resistant tobromodeoxyuridine (BrdU) and a cell lacking HGPRT is resistant to 6-thioguanine (6-TG) and 8-azaguanine . Thus selection with one of the latter two drugs, followed by HAT medium, will yieldrevertant colonies. [cite web|url=http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=21144|title=evidence for gene silencing by endogenous methylation|accessdate=05-07-07]Applications
HAT medium is often used for preparation of
monoclonal antibodies . This process is called Hybridoma technology. Laboratory animals (eg. mice) are first exposed to anantigen to which we are interested in isolating anantibody against. Once splenocytes are isolated from the mammal, theB cells are fused with HGPRT negative,immortalized myeloma cells usingpolyethylene glycol or theSendai virus . Fused cells are incubated in the "HAT medium".Aminopterin in the medium blocks the de novo pathway. Hence, unfused myeloma cells die, as they cannot produce nucleotides by de novo or salvage pathway. Unfused B cells die as they have a short life span. In this way, only the B cell-myeloma hybrids survive. These cells produceantibodies (a property of B cells) and are immortal (a property of myeloma cells).The incubated medium is then diluted into multiwell plates to such an extent that each well contains only 1 cell. Then thesupernatant in each well can be checked for desired antibody. Since the antibodies in a well are produced by the same B cell, they will be directed towards the sameepitope , and are known asmonoclonal antibodies .The production of monoclonal anti-bodies was first invented by Cesar Milstein, George Kohler and Niels Kaj Jerne in 1975
References
Wikimedia Foundation. 2010.