Lodish H. - Molecular Cell Biology (5ed, Freeman, 2003) (794361), страница 102
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Each hybridoma produces a singleantibody. Step 3 : Testing of individual clones identifies thosethat recognize antigen X. After a hybridoma that produces adesired antibody has been identified, the clone can becultured to yield large amounts of that antibody.one type of monoclonal antibody from blood is not feasible, in part because the concentration of any given antibodyis quite low.Because of their limited life span, primary cultures ofnormal B lymphocytes are of limited usefulness for the production of monoclonal antibody.
Thus the first step in producing a monoclonal antibody is to generate immortal,antibody-producing cells. This immortality is achieved byfusing normal B lymphocytes from an immunized animalwith transformed, immortal lymphocytes called myelomacells. During cell fusion, the plasma membranes of two cellsfuse together, allowing their cytosols and organelles to intermingle. Treatment with certain viral glycoproteins or thechemical polyethylene glycol promotes cell fusion.
Some ofthe fused cells can undergo division and their nuclei eventually coalesce, producing viable hybrid cells with a singlenucleus that contains chromosomes from both “parents.”The fusion of two cells that are genetically different canyield a hybrid cell with novel characteristics. For instance,the fusion of a myeloma cell with a normal antibodyproducing cell from a rat or mouse spleen yields a hybridthat proliferates into a clone called a hybridoma. Likemyeloma cells, hybridoma cells grow rapidly and are immortal. Each hybridoma produces the monoclonal antibodyencoded by its B-lymphocyte parent.The second step in this procedure for producing monoclonal antibody is to separate, or select, the hybridoma cellsfrom the unfused parental cells and the self-fused cells generated by the fusion reaction. This selection is usually performed by incubating the mixture of cells in a specialculture medium, called selection medium, that permits thegrowth of only the hybridoma cells because of their novelcharacteristics.
Such a selection is readily performed if themyeloma cells used for the fusion carry a mutation thatblocks a metabolic pathway and renders them, but not theirlymphocyte fusion partners that do not have the mutation,sensitive to killing by the selection medium. In the immortal hybrid cells, the functional gene from the lymphocytecan supply the gene product missing because of the mutation in the myeloma cell, and thus the hybridoma cells butnot the myeloma cells, will be able to grow in the selectionmedium.
Because the lymphocytes used in the fusion arenot immortalized and do not divide rapidly, only the hybridoma cells will proliferate rapidly in the selectionmedium and can thus be readily isolated from the initialmixture of cells.Figure 6-38 depicts the general procedure for generating and selecting hybridomas. In this case, normal B lymphocytes are fused with myeloma cells that cannot growin HAT medium, the most common selection medium usedin the production of hybridomas. Only the myelomalymphocyte hybrids can survive and grow for an extendedperiod in HAT medium for reasons described shortly.Thus, this selection medium permits the separation of hybridoma cells from both types of parental cells and any6.7 • Growth and Use of Cultured Cellsself-fused cells. Finally, each selected hybridoma is thentested for the production of the desired antibody; anyclone producing that antibody is then grown in large cultures, from which a substantial quantity of pure monoclonal antibody can be obtained.Monoclonal antibodies are commonly employedin affinity chromatography to isolate and purifyproteins from complex mixtures (see Figure 3-34c).They can also be used to label and thus locate a particularprotein in specific cells of an organ and within culturedcells with the use of immunofluorescence microscopy techniques (see Figures 6-26a and 6-27) or in specific cell fractions with the use of immunoblotting (see Figure 3-35).Monoclonal antibodies also have become important diagnostic and therapeutic tools in medicine.
For example,monoclonal antibodies that bind to and inactivate toxicproteins (toxins) secreted by bacterial pathogens are usedto treat diseases caused by these pathogens. Other monoclonal antibodies are specific for cell-surface proteins expressed by certain types of tumor cells; chemical complexesof such monoclonal antibodies with toxic drugs or simplythe antibodies themselves have been developed for cancerchemotherapy.
❚239HAT Medium Is Commonly Used to IsolateHybrid CellsThe principles underlying HAT selection are important notonly for understanding how hybridoma cells are isolated butalso for understanding several other frequently used selectionmethods, including selection of the ES cells used in generating knockout mice (Chapter 9).
HAT medium contains hypoxanthine (a purine), aminopterin, and thymidine. Mostanimal cells can synthesize the purine and pyrimidine nucleotides from simpler carbon and nitrogen compounds (Figure 6-39, top). The folic acid antagonists amethopterin andaminopterin interfere with the donation of methyl andformyl groups by tetrahydrofolic acid in the early stages ofthe synthesis of glycine, purine nucleoside monophosphates,and thymidine monophosphate.
These drugs are called antifolates because they block reactions of tetrahydrofolate, anactive form of folic acid.Many cells, however, are resistant to antifolates becausethey contain enzymes that can synthesize the necessary nucleotides from purine bases and thymidine (Figure 6-39, bottom). Two key enzymes in these nucleotide salvage pathwaysare thymidine kinase (TK) and hypoxanthine-guaninephosphoribosyl transferase (HGPRT). Cells that producethese enzymes can grow on HAT medium, which supplies aDe novosynthesis ofpurine nucleotidesPRPP (5-Phosphoribosyl-1-pyrophosphate)Blocked by antifolatesDe novosynthesis ofTMPDeoxyuridylate (dUMP)CHO from tetrahydrofolateBlocked by antifolatesBlocked by antifolatesCHO from tetrahydrofolateNucleicacidsGuanylate (GMP)PRPPInosinate (IMP)HGPRT(hypoxanthineguaninephosphoribosyltransferase)GuanineAdenylate (AMP)NucleicacidsAPRT(adeninephosphoribosyltransferase)PRPPHGPRTPRPPHypoxanthineAdenineCH3 fromtetrahydrofolateThymidylate (TMP)TK(thymidine kinase)ThymidineSalvage pathways▲ FIGURE 6-39 De novo and salvage pathways fornucleotide synthesis.
Animal cells can synthesize purinenucleotides (AMP, GMP, IMP) and thymidylate (TMP) fromsimpler compounds by de novo pathways (blue). They require thetransfer of a methyl or formyl (“CHO”) group from an activatedform of tetrahydrofolate (e.g., N 5,N 10-methylenetetrahydrofolate),as shown in the upper part of the diagram. Antifolates, suchas aminopterin and amethopterin, block the reactivation oftetrahydrofolate, preventing purine and thymidylate synthesis.Many animal cells can also use salvage pathways (red) toincorporate purine bases or nucleosides and thymidine.
If theseprecursors are present in the medium, normal cells will groweven in the presence of antifolates. Cultured cells lacking one ofthe enzymes—HGPRT, APRT, or TK—of the salvage pathwayswill not survive in media containing antifolates.240CHAPTER 6 • Integrating Cells into Tissuessalvageable purine and thymidine, whereas those lacking oneof them cannot.Cells with a TK mutation that prevents the productionof the functional TK enzyme can be isolated because suchcells are resistant to the otherwise toxic thymidine analog5-bromodeoxyuridine.
Cells containing TK convert thiscompound into 5-bromodeoxyuridine monophosphate,which is then converted into a nucleoside triphosphate byother enzymes. The triphosphate analog is incorporated byDNA polymerase into DNA, where it exerts its toxic effects.This pathway is blocked in TK mutants, and thus they areresistant to the toxic effects of 5-bromodeoxyuridine. Similarly, cells lacking the HGPRT enzyme, such as the HGPRTmyeloma cell lines used in producing hybridomas, can be isolated because they are resistant to the otherwise toxic guanine analog 6-thioguanine.Normal cells can grow in HAT medium because eventhough the aminopterin in the medium blocks de novo synthesis of purines and TMP, the thymidine in the medium istransported into the cell and converted into TMP by TK andthe hypoxanthine is transported and converted into usablepurines by HGPRT.
On the other hand, neither TK norHGPRT cells can grow in HAT medium because each lacksan enzyme of the salvage pathway. However, hybrids formedby the fusion of these two mutants will carry a normal TKgene from the HGPRT parent and a normal HGPRT genefrom the TK parent. The hybrids will thus produce bothfunctional salvage-pathway enzymes and will grow on HATmedium.KEY CONCEPTS OF SECTION 6.7Growth and Use of Cultured CellsGrowth of vertebrate cells in culture requires rich media containing essential amino acids, vitamins, fatty acids,and peptide or protein growth factors; the last are frequently provided by serum.■Most cultured vertebrate cells will grow only when attached to a negatively charged substratum that is coatedwith components of the extracellular matrix.■Primary cells, which are derived directly from animal tissue, have limited growth potential in culture and may giverise to a cell strain.
Transformed cells, which are derivedfrom animal tumors or arise spontaneously from primarycells, grow indefinitely in culture, forming cell lines (seeFigure 6-37).■The fusion of an immortal myeloma cell and a singleB lymphocyte yields a hybrid cell that can proliferateindefinitely, forming a clone called a hybridoma (seeFigure 6-38). Because each individual B lymphocyteproduces antibodies specific for one antigenic determinant (epitope), a hybridoma produces only the mono-■clonal antibody synthesized by its original B-lymphocyteparental cell.HAT medium is commonly used to isolated hybridomacells and other types of hybrid cells.■PERSPECTIVES FOR THE FUTUREA deeper understanding of the integration of cells into tissuesin complex organisms will draw on insights and techniquesfrom virtually all subdisciplines of molecular cell biology: biochemistry, biophysics, microscopy, genetics, genomics, proteomics, and developmental biology.
An important set ofquestions for the future deals with the mechanisms by whichcells detect mechanical forces on them and the extracellularmatrix, as well as the influence of their three-dimensionalarrangements and interactions. A related question is how thisinformation is used to control cell and tissue structure andfunction. Shear stresses can induce distinct patterns of gene expression and cell growth and can greatly alter cell metabolismand responses to extracellular stimuli. Future research shouldgive us a far more sophisticated understanding of the roles ofthe three-dimensional organization of cells and ECM components in controlling the structures and activities of tissues.Numerous questions relate to intracellular signaling fromCAMs and adhesion receptors. Such signaling must be integrated with other cellular signaling pathways that are activated by various external signals (e.g., growth factors) sothat the cell responds appropriately and in a single coordinated fashion to many different simultaneous internal andexternal stimuli.
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