HAT Medium is a selection medium for mammalian cell culture, which relies on the combination of aminopterin, a drug that acts as a powerful folate metabolism inhibitor by inhibiting dihydrofolate reductase

Dihydrofolate reductase

- Function :Dihydrofolate reductase converts dihydrofolate into tetrahydrofolate, a methyl group shuttle required for the de novo synthesis of purines, thymidylic acid, and certain amino acids...

, with hypoxanthine

Hypoxanthine

Hypoxanthine is a naturally occurring purine derivative. It is occasionally found as a constituent of nucleic acids where it is present in the anticodon of tRNA in the form of its nucleoside inosine. It has a tautomer known as 6-Hydroxypurine. Hypoxanthine is a necessary additive in certain cell,...

 (a purine

Purine

A purine is a heterocyclic aromatic organic compound, consisting of a pyrimidine ring fused to an imidazole ring. Purines, including substituted purines and their tautomers, are the most widely distributed kind of nitrogen-containing heterocycle in nature....

 derivative) and thymidine

Thymidine

Thymidine is a chemical compound, more precisely a pyrimidine deoxynucleoside. Deoxythymidine is the DNA nucleoside T, which pairs with deoxyadenosine in double-stranded DNA...

 (a deoxynucleoside

Nucleoside

Nucleosides are glycosylamines consisting of a nucleobase bound to a ribose or deoxyribose sugar via a beta-glycosidic linkage...

) which are intermediates in DNA synthesis

DNA synthesis

DNA synthesis commonly refers to:*DNA replication - DNA biosynthesis *Polymerase chain reaction - enzymatic DNA synthesis *Oligonucleotide synthesis - chemical synthesis of nucleic acids...

. The trick is that aminopterin blocks DNA de novo synthesis

De novo synthesis

De novo synthesis refers to the synthesis of complex molecules from simple molecules such as sugars or amino acids, as opposed to their being recycled after partial degradation. For example, nucleotides are not needed in the diet as they can be constructed from small precursor molecules such as...

, which is absolutely required for cell division

Cell division

Cell division is the process by which a parent cell divides into two or more daughter cells . Cell division is usually a small segment of a larger cell cycle. This type of cell division in eukaryotes is known as mitosis, and leaves the daughter cell capable of dividing again. The corresponding sort...

 to proceed, but hypoxanthine and thymidine provide cells with the raw material to evade the blockage (the "salvage pathway"), provided that they have the right enzymes, which means having functioning copies of the genes

Gênes

Gênes is the name of a département of the First French Empire in present Italy, named after the city of Genoa. It was formed in 1805, when Napoleon Bonaparte occupied the Republic of Genoa. Its capital was Genoa, and it was divided in the arrondissements of Genoa, Bobbio, Novi Ligure, Tortona and...

 that encode them.

The enzyme dihydrofolate reductase

Dihydrofolate reductase

- Function :Dihydrofolate reductase converts dihydrofolate into tetrahydrofolate, a methyl group shuttle required for the de novo synthesis of purines, thymidylic acid, and certain amino acids...

, which produces tetrahydrofolate (THF) by the reduction of dihydrofolate, is specifically blocked by aminopterin. THF, acting in association with specific protein

Protein

Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...

s, can receive single carbon units that are then transferred to specific targets.

One of the important targets for cellular reproduction is thymidylate synthase

Thymidylate synthase

Thymidylate synthetase is the enzyme used to generate thymidine monophosphate , which is subsequently phosphorylated to thymidine triphosphate for use in DNA synthesis and repair....

, which creates thymidine monophosphate

Thymidine monophosphate

Thymidine monophosphate, also known as 5'-thymidylate, thymidylate, or TMP, is a nucleotide that is used as a monomer in DNA. It is an ester of phosphoric acid with the nucleoside thymidine. TMP consists of a phosphate group, the pentose sugar deoxyribose, and the nucleobase thymine...

 (TMP) from deoxyuridine monophosphate

Deoxyuridine monophosphate

Deoxyuridine monophosphate is a deoxynucleotide.It is an intermediate in the metabolism of deoxyribonucleotides.-See also:* uridine monophosphate* DCMP deaminase...

 (dUMP). By additional phosphorylation

Phosphorylation

Phosphorylation is the addition of a phosphate group to a protein or other organic molecule. Phosphorylation activates or deactivates many protein enzymes....

 reactions, TMP can be used to make thymidine triphosphate

Thymidine triphosphate

5-Methyluridine triphosphate or m5UTP is one of five nucleoside triphosphates. It is the ribonucleoside triphosphate of thymidine, but the 5-methyluridine nomenclature is used because the term thymidine triphosphate is used for the deoxyribonucleoside by convention.-References:...

 (TTP), one of the four nucleotide precursors that are used by DNA polymerases to create DNA

DNA

Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...

. 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 that thymidine

Thymidine

Thymidine is a chemical compound, more precisely a pyrimidine deoxynucleoside. Deoxythymidine is the DNA nucleoside T, which pairs with deoxyadenosine in double-stranded DNA...

 present in HAT medium can be absorbed by the cells and phosphorylated by thymidine kinase

Thymidine kinase

Thymidine kinase is an enzyme, a phosphotransferase : 2'-deoxythymidine kinase, ATP-thymidine 5'-phosphotransferase, . It can be found in most living cells. It is present in two forms in mammalian cells, TK1 and TK2...

 (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

Hypoxanthine-guanine phosphoribosyltransferase

Hypoxanthine-guanine phosphoribosyltransferase is an enzyme encoded in humans by the HPRT1 gene.HGPRT is a transferase that catalyzes conversion of hypoxanthine to inosine monophosphate and guanine to guanosine monophosphate. This reaction transfers the 5-phosphoribosyl group from...

 (HGPRT) reacts hypoxanthine absorbed from the medium with PRPP, liberating pyrophosphate

Pyrophosphate

In chemistry, the anion, the salts, and the esters of pyrophosphoric acid are called pyrophosphates. Any salt or ester containing two phosphate groups is called a diphosphate. As a food additive, diphosphates are known as E450.- Chemistry :...

, to produce IMP by a salvage pathway.

Therefore, the use of HAT medium for cell culture is a form of artificial selection

Artificial selection

Artificial selection describes intentional breeding for certain traits, or combination of traits. The term was utilized by Charles Darwin in contrast to natural selection, in which the differential reproduction of organisms with certain traits is attributed to improved survival or reproductive...

 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 to bromodeoxyuridine

Bromodeoxyuridine

Bromodeoxyuridine is a synthetic nucleoside that is an analogue of thymidine. BrdU is commonly used in the detection of proliferating cells in living tissues....

 (BrdU) and a cell lacking HGPRT is resistant to 6-thioguanine (6-TG) and 8-azaguanine

8-Azaguanine

8-Azaguanine is a purine analog with the chemical formula C4H4N6O. It has been widely studied for its biological activity. It shows antineoplastic activity and has been used in the treatment of acute leukemia.-Use in chemotherapy:...

. Thus, selection with one of the latter two drugs, followed by HAT medium, will yield revertant

Revertant

In microbial genetics, a revertant is a mutant that has reverted to its former genotype or to the original phenotype by means of a suppressor mutation, or else by compensatory mutation somewhere in the gene ....

 colonies.

Applications

HAT medium is often used for preparation of monoclonal antibodies

Monoclonal antibodies

Monoclonal antibodies are monospecific antibodies that are the same because they are made by identical immune cells that are all clones of a unique parent cell....

. This process is called Hybridoma technology

Hybridoma

Hybridoma technology is a technology of forming hybrid cell lines by fusing a specific antibody-producing B cell with a myeloma cell that is selected for its ability to grow in tissue culture and for an absence of antibody chain synthesis...

. Laboratory animals (e.g., mice) are first exposed to an antigen

Antigen

An antigen is a foreign molecule that, when introduced into the body, triggers the production of an antibody by the immune system. The immune system will then kill or neutralize the antigen that is recognized as a foreign and potentially harmful invader. These invaders can be molecules such as...

 against which we are interested in isolating an antibody. Once splenocytes are isolated from the mammal, the B cells are fused with HPRT negative, immortalized myeloma cells using polyethylene glycol

Polyethylene glycol

Polyethylene glycol is a polyether compound with many applications from industrial manufacturing to medicine. It has also been known as polyethylene oxide or polyoxyethylene , depending on its molecular weight, and under the tradename Carbowax.-Available forms:PEG, PEO, or POE refers to an...

 or the Sendai virus

Sendai virus

Sendai virus , also known as murine parainfluenza virus type 1 or hemagglutinating virus of Japan , is a negative sense, single-stranded RNA virus of the Paramyxoviridae family, a group of viruses featuring, notably, the Morbillivirus and Rubulavirus genera...

. Fused cells are incubated in the HAT medium. Aminopterin

Aminopterin

Aminopterin , a 4-amino analog of folic acid, is an antineoplastic drug with immunosuppressive properties used in chemotherapy. Aminopterin is a synthetic derivative of pterin. Aminopterin works as an enzyme inhibitor by competing for the folate binding site of the enzyme dihydrofolate reductase...

 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 lifespan. In this way, only the B cell-myeloma hybrids survive. These cells produce antibodies (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 the supernatant 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 same epitope

Epitope

An epitope, also known as antigenic determinant, is the part of an antigen that is recognized by the immune system, specifically by antibodies, B cells, or T cells. The part of an antibody that recognizes the epitope is called a paratope...

, and are known as monoclonal antibodies

Monoclonal antibodies

Monoclonal antibodies are monospecific antibodies that are the same because they are made by identical immune cells that are all clones of a unique parent cell....

.

The production of monoclonal anti-bodies was first invented by Cesar Milstein

César Milstein

César Milstein FRS was an Argentine biochemist in the field of antibody research. Milstein shared the Nobel Prize in Physiology or Medicine in 1984 with Niels K. Jerne and Georges Köhler.-Biography:...

, Georges J. F. Köhler

Georges J. F. Köhler

-External links:* http://www.nobel.se/medicine/laureates/1984/...

 and Niels Kaj Jerne

Niels Kaj Jerne

Niels Kaj Jerne, FRS was a Danish immunologist. He was awarded the Nobel Prize in Physiology or Medicine in 1984. The citation read "For theories concerning the specificity in development and control of the immune system and the discovery of the principle for production of monoclonal antibodies"....

in 1975.