User guide

Genomic amplification by MTX addition
Introduction
Methotrexate (MTX) is a folic acid antagonist that is actively transported into cells
by the folate transporter. In the cell, it is converted to a high molecular weight
polyglutamate metabolite by folylpolyglutamate synthase, which binds to DHFR
and inhibits its activity.
If MTX is present in the medium, cells compensate by increasing the DHFR copy
number in the genome to overcome inhibition by MTX. Since the gene of interest
is integrated into the same genetic locus as DHFR, the gene of interest is amplified
as well, leading to increased production of the protein of interest (Kaufman et al.,
1985; Tanaka et al., 2002). MTX (as methotrexate hydrate) is available from Sigma
(10 mg, Sigma Cat. no. A6770).
CAUTION!
MTX is toxic to the skin, eyes, and respiratory system. Wear suitable protective
clothing, gloves, and eye and face protection when working with MTX. Refer to
the product MSDS for complete precautions.
Preparing
1 mM MTX
To prepare a 1 mM MTX stock solution:
1. Dissolve 10 mg MTX in 22 mL of PBS.
2. Filter-sterilize the solution through a 0.22 µm filter.
3. Store in 250 µL aliquots at –20°C.
Preparing media
with MTX
• To make complete CD OptiCHO
™
Medium containing MTX, use complete
CD OptiCHO
™
Medium (prepared to contain L-glutamine) plus the required
concentration of MTX.
• Do not add Geneticin
®
reagent to the complete CD OptiCHO
™
Medium as the
cells transfected with only the pOptiVEC
™
construct do not express resistance to
Geneticin
®
Selective Antibiotic.
• Using the sterile, 1 mM MTX stock solution (prepared as described above),
prepare media containing the appropriate concentration of MTX.
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