What is MTX amplification?
A method combining the use of a destabilized dihydrofolate reductase (DHFR) selection marker with methotrexate (MTX) amplification to generate high-expressing cells is described here. Low concentrations of MTX are then used to amplify the transfected genes for increased protein expression.
How does gene amplification occur?
Gene amplification refers to an increase in the number of copies of the same gene rather than to an increase in its rate of transcription. It results from gene duplication that has been repeated many times over, producing from 100 to 1000 copies of the gene.
What is the function of dihydrofolate reductase?
Dihydrofolate reductase (DHFR) catalyzes the reduction of dihydrofolate to tetrahydrofolate (THF). THF is needed for the action of folate-dependent enzymes and is thus essential for DNA synthesis and methylation.
How does resistance to methotrexate develop?
In experimental systems, resistance to MTX can occur through a variety of mechanisms, including impaired transport of drug into the cell via the RFC, an increase in DHFR due to gene amplification or increased transcription, and diminished intracellular retention secondary to decreased polyglutamylation (5) .
Why is methotrexate used in cell culture?
Methotrexate is a drug which is similar (i.e., an analog) to folate. MTX binds to DHFR, thereby inhibiting the production of tetrahydrofolate. With insufficient levels of DHFR, cells are deprived of nucleoside precursors (hypoxanthine and thymidine) and die.
What is the function of gene amplification?
What is the function of reductase?
Reductase enzymes belong to the E.C. 1 class of oxidoreductases and catalyze reduction reactions. A reduction reaction involves the gain of electrons; it is usually coupled to oxidation and termed a redox reaction. Reductases lower the activation energy needed for redox reactions to occur.
Do humans have dihydrofolate?
In humans, the DHFR enzyme is encoded by the DHFR gene. It is found in the q11→q22 region of chromosome 5. Bacterial species possess distinct DHFR enzymes (based on their pattern of binding diaminoheterocyclic molecules), but mammalian DHFRs are highly similar.
What is the mechanism of action of methotrexate?
Methotrexate inhibits dihydrofolate reductase, preventing the reduction of dihydrobiopterin (BH2) to tetrahydrobiopterin (BH4), leading to nitric oxide synthase uncoupling and increased sensitivity of T cells to apoptosis, thereby diminishing immune responses.
Which chemo drugs are cell cycle specific?
Antitumor Antibiotics These drugs act during multiple phases of the cell cycle and are considered cell-cycle specific. There are several types of antitumor antibiotics: Anthracyclines: Doxorubicin, Daunorubicin, Epirubicin, Mitoxantrone, and Idarubicin. Chromomycins: Dactinomycin and Plicamycin.
