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The enzyme is inhibited by methotrexate.
Sensitivity to methotrexate can be overcome if the cell produces excess dihydrofolate reductase, and as the methotrexate concen tration is increased over time, the dihydrofolate reductase gene in cultured cells is amplified.
Selective agent Action of selective agent Marker gene Action of marker gene protein Xyl-A Damages DNA Adenine deaminase (ada) Deaminates Xyl-A Blasticidin S Inhibits protein synthesis Blasticidin S deaminases (Bsr, BSD) Deaminates blasticidin S Bleomycin Breaks DNA strands Bleomycin-binding protein (Ble) Binds to bleomycin G-418 (Geneticin) Inhibits protein synthesis Neomycin phosphotransferase (neo) Phosphorylates G-418 Histidinol Produces cytotoxic efects Histidinol dehydrogenase (hisD) Oxidizes histidinol to histidine Hygromycin B Inhibits protein synthesis Hygromycin B phosphotransferase (Hph) Phosphorylates hygromycin B MSX Inhibits glutamine synthesis Glutamine synthetase (GS) Cells that produce excess glutamine synthetase survive MTX Inhibits DNA synthesis Dihydrofolate reductase (dhfr) Cells that produce excess dihydrofolate reductase survive PALA Inhibits purine synthesis Cytosine deaminase (codA) Lowers cytosine levels in the medium by converting cytosine to uracil Puromycin Inhibits protein synthesis Puromycin N-acetyltransferase (Pac) Acetylates puromycin MSX, methionine sulfoximine; MTX, methotrexate; PALA, N(phosphoacetyl)laspartate; XylA, 9کdxylofuranosyl adenine.
reductase-methotrexate protocol entails transfecting dihydrofolate reduc tasedeficient cells with an expression vector carrying a dihydrofolate re ductase gene as the selectable marker gene and treating the cells with methotrexate.
After the initial selection of transfected cells, the concentra tion of methotrexate is gradually increased, and eventually cells with very high copy numbers of the expression vector are selected.
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