HAT Medium (

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. Hyp ...

aminopterin Aminopterin (or 4-aminopteroic acid), the 4–amino derivative of folic acid, is an antineoplastic drug with immunosuppressive properties often used in chemotherapy. Aminopterin is a synthetic derivative of pterin. Aminopterin works as an enzyme ...

thymidine Thymidine (nucleoside#List of nucleosides and corresponding nucleobases, symbol dT or dThd), also known as deoxythymidine, deoxyribosylthymine, or thymine deoxyriboside, is a pyrimidine nucleoside, deoxynucleoside. Deoxythymidine is the DNA nuc ...

medium) is a selection medium for mammalian cell culture, which relies on the combination of

, a drug that acts as a powerful

folate Folate, also known as vitamin B9 and folacin, is one of the B vitamins. Manufactured folic acid, which is converted into folate by the body, is used as a dietary supplement and in food fortification as it is more stable during processing and ...

metabolism inhibitor by inhibiting dihydrofolate reductase, with

purine Purine is a heterocyclic aromatic organic compound that consists of two rings (pyrimidine and imidazole) fused together. It is water-soluble. Purine also gives its name to the wider class of molecules, purines, which include substituted puri ...

derivative) and

(a deoxy

nucleoside Nucleosides are glycosylamines that can be thought of as nucleotides without a phosphate group. A nucleoside consists simply of a nucleobase (also termed a nitrogenous base) and a five-carbon sugar (ribose or 2'-deoxyribose) whereas a nucleotid ...

) which are intermediates in DNA synthesis. The trick is that aminopterin blocks DNA ''de novo'' synthesis, which is absolutely required for

cell division Cell division is the process by which a parent cell (biology), cell divides into two daughter cells. Cell division usually occurs as part of a larger cell cycle in which the cell grows and replicates its chromosome(s) before dividing. In eukar ...

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 An enzyme () is a protein that acts as a biological catalyst by accelerating chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as pro ...

, which means having functioning copies of the

genes In biology, the word gene has two meanings. The Mendelian gene is a basic unit of heredity. The molecular gene is a sequence of nucleotides in DNA that is transcribed to produce a functional RNA. There are two types of molecular genes: protei ...

that encode them. The enzyme dihydrofolate reductase, which produces tetrahydrofolate (THF) by the reduction of dihydrofolate, is specifically blocked by aminopterin. THF, acting in association with specific

protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residue (biochemistry), residues. Proteins perform a vast array of functions within organisms, including Enzyme catalysis, catalysing metab ...

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 (TS) () is an enzyme that catalyzes the conversion of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP). Thymidine is one of the nucleotides in DNA. With inhibition of TS, an imbalance of deoxynucleot ...

, which creates thymidine monophosphate (TMP) from

deoxyuridine monophosphate Deoxyuridine monophosphate (dUMP), also known as deoxyuridylic acid or deoxyuridylate in its conjugate acid and conjugate base forms, respectively, is a deoxynucleotide. It is an intermediate in the metabolism of deoxyribonucleotides. Biosynthes ...

(dUMP). By additional

phosphorylation In biochemistry, phosphorylation is described as the "transfer of a phosphate group" from a donor to an acceptor. A common phosphorylating agent (phosphate donor) is ATP and a common family of acceptor are alcohols: : This equation can be writ ...

reactions, TMP can be used to make

thymidine triphosphate Thymidine triphosphate (TTP), also called deoxythymidine triphosphate (dTTP), is one of the four nucleoside triphosphates that are used in the ''in vivo'' synthesis of DNA. Unlike the other deoxyribonucleoside triphosphates, thymidine triphosphate ...

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

DNA Deoxyribonucleic acid (; DNA) is a polymer composed of two polynucleotide chains that coil around each other to form a double helix. The polymer carries genetic instructions for the development, functioning, growth and reproduction of al ...

. 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 the

present in the HAT medium that can be absorbed by the cells and phosphorylated by thymidine kinase (TK) into TMP. The synthesis of IMP, (precursor to GMP and GTP, and to AMP and ATP) also requires THF, and also can be bypassed. In this case hypoxanthine-guanine phosphoribosyltransferase (HGPRT) reacts hypoxanthine absorbed from the medium with PRPP, liberating

pyrophosphate In chemistry, pyrophosphates are phosphorus oxyanions that contain two phosphorus atoms in a linkage. A number of pyrophosphate salts exist, such as disodium pyrophosphate () and tetrasodium pyrophosphate (), among others. Often pyrophosphates a ...

, to produce IMP by a salvage pathway. Therefore, the use of HAT medium for cell culture is a form of

artificial selection Selective breeding (also called artificial selection) is the process by which humans use animal breeding and plant breeding to selectively develop particular phenotypic traits (characteristics) by choosing which typically animal or plant ...

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 (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 yield revertant colonies.{{cite journal, title=evidence for gene silencing by endogenous methylation, pmc=21144 , pmid=9671746 , volume=95, journal=Proc Natl Acad Sci U S A, pages=8727–32 , last1 = Holliday , first1 = R , last2 = Ho , first2 = T , year=1998 , issue=15 , doi=10.1073/pnas.95.15.8727, bibcode=1998PNAS...95.8727H , doi-access=free

Applications

HAT medium is used for preparation of

monoclonal antibodies A monoclonal antibody (mAb, more rarely called moAb) is an antibody produced from a Lineage (evolution), cell lineage made by cloning a unique white blood cell. All subsequent antibodies derived this way trace back to a unique parent cell. Mon ...

. This process is called hybridoma technology. Laboratory animals (e.g., mice) are first exposed to an

antigen In immunology, an antigen (Ag) is a molecule, moiety, foreign particulate matter, or an allergen, such as pollen, that can bind to a specific antibody or T-cell receptor. The presence of antigens in the body may trigger an immune response. ...

against which we are interested in isolating an

antibody An antibody (Ab) or immunoglobulin (Ig) is a large, Y-shaped protein belonging to the immunoglobulin superfamily which is used by the immune system to identify and neutralize antigens such as pathogenic bacteria, bacteria and viruses, includin ...

. Once splenocytes are isolated from the mammal, the

B cells B cells, also known as B lymphocytes, are a type of the lymphocyte subtype. They function in the humoral immunity component of the adaptive immune system. B cells produce antibody molecules which may be either secreted or inserted into the plasm ...

are fused with HGPRT negative, immortalized

myeloma Multiple myeloma (MM), also known as plasma cell myeloma and simply myeloma, is a cancer of plasma cells, a type of white blood cell that normally produces antibodies. Often, no symptoms are noticed initially. As it progresses, bone pain, a ...

cells using

polyethylene glycol Polyethylene glycol (PEG; ) is a polyether compound derived from petroleum with many applications, from industrial manufacturing to medicine. PEG is also known as polyethylene oxide (PEO) or polyoxyethylene (POE), depending on its molecular wei ...

or the

Sendai virus ''Murine respirovirus'', formerly ''Sendai virus'' (SeV) and previously also known as murine parainfluenza virus type 1 or hemagglutinating virus of Japan (HVJ), is an Viral envelope, enveloped, 150-200 nm–diameter, negative sense, single ...

. Fused cells are incubated in the ''HAT medium''.

Aminopterin Aminopterin (or 4-aminopteroic acid), the 4–amino derivative of folic acid, is an antineoplastic drug with immunosuppressive properties often used in chemotherapy. Aminopterin is a synthetic derivative of pterin. Aminopterin works as an enzyme ...

in the medium blocks the ''de novo'' pathway. Hence, unfused myeloma cells die, as they cannot produce nucleotides by the ''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 An antibody (Ab) or immunoglobulin (Ig) is a large, Y-shaped protein belonging to the immunoglobulin superfamily which is used by the immune system to identify and neutralize antigens such as bacteria and viruses, including those that caus ...

(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 an aqueous solution, precipitation is the "sedimentation of a solid material (a precipitate) from a liquid solution". The solid formed is called the precipitate. In case of an inorganic chemical reaction leading to precipitation, the chemic ...

in each well can be checked for the desired antibody. Since the antibodies in a well are produced by the same B cell, they will be directed towards the same

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 binds to the epitope is called a paratope. Although e ...

, and are known as

. The production of monoclonal antibodies was first invented by César Milstein and Georges J. F. Köhler, which earned them the 1984 Nobel Prize in Physiology or Medicine, shared with Niels Kaj Jerne.

References

Cell culture media