User guide

Selecting stable transfectants for two-subunit protein
expression
Introduction
To obtain cell lines that produce high levels of your protein, first select for a pool
of stably-transfected cells, in which the linearized pOptiVEC
™
and pcDNA
™
3.3
constructs have integrated into the host cell genome. Perform the selection using
complete CD OptiCHO
™
Medium containing 500 µg/mL of Geneticin
®
Selective
Antibiotic. Note that only cells that have been transfected with pOptiVEC
™
and
pcDNA
™
3.3 constructs can be propagated in CD OptiCHO
™
Medium + Geneticin
®
reagent, because untransfected CHO DG44 Cells (cGMP banked) lack APH and
DHFR activities (see below).
Geneticin

Selective Antibiotic
Geneticin
®
Selective Antibiotic (G-418) blocks protein synthesis in mammalian
cells by interfering with ribosomal function. It is an aminoglycoside, similar in
structure to neomycin, gentamycin, and kanamycin. Expression in mammalian
cells of the bacterial gene (APH), derived from Tn5, results in detoxification of
Geneticin
®
reagent (Southern & Berg, 1982).
Calculate the concentration based on the amount of active drug. Cells will divide
once or twice in the presence of lethal doses of Geneticin
®
reagent, so the effects of
the drug take several days to become apparent. Complete selection can take up to
two weeks of growth in selective medium.
Note
Development work with this kit used 500 µg/mL of Geneticin
®
reagent; however,
because different transfected cells may exhibit different Geneticin
®
Selective
Antibiotic sensitivity, we recommend that you conduct a kill-curve study to
establish the ideal concentration of Geneticin
®
reagent for using with your cells. See
page
19, Establishing Sensitivity to Geneticin
®
Selective Antibiotic (G-418), for a
kill-curve study protocol.
DHFR
DHFR catalyzes the reduction of 5, 6-dihydrofolate to 5, 6, 7, 8-tetrahydrofolate,
which is essential for DNA synthesis. CHO-derived CHO DG44 Cells (cGMP
banked) lack DHFR activity, and they must be propagated in medium containing
the purine precursors hypoxanthine and thymidine (HT) unless the cells are stably
transfected with a vector that expresses DHFR.
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