User guide

Description of the system
Freedom
™
DG44 Kit
The Freedom
™
DG44 Kit is designed for easy cloning and expression of
recombinant proteins in dihydrofolate reductase (DHFR)-deficient, Chinese
hamster ovary (CHO)-derived CHO DG44 Cells (cGMP banked) in suspension
culture. The Freedom
™
DG44 Kit provides reagents for:
• cloning the gene(s) that encode a single or two-subunit protein
• transfecting the DNA constructs into CHO DG44 Cells (cGMP banked) with
high efficiency
•
generating stable cell lines that produce your protein of interest
Components of the
Freedom
™
DG44 Kit
• pOptiVEC
™
-TOPO
®
TA Cloning Kit: A TOPO
®
-adapted bicistronic plasmid
and reagents for cloning a PCR product containing a mammalian secretion
signal and each subunit, separately, of your protein of interest. See the next
page for more information.
• pcDNA
™
3.3-TOPO
®
TA Cloning Kit: A TOPO
®
-adapted plasmid and reagents
for cloning a PCR product containing a mammalian secretion signal and each
subunit, separately, of your protein of interest. See the next page for more
information.
• CHO DG44 Cells (cGMP banked): cGMP banked, DHFR-negative,
CHO-derived cells adapted to high density, serum-free suspension culture in
CD DG44 Medium that are capable of producing high levels of secreted,
recombinant protein. See page 10 for more information.
• CD DG44 Medium: Defined, serum-free medium supplemented with
hypoxanthine and thymidine to allow growth of DHFR-negative CHO DG44
Cells (cGMP banked). See page 11 for more information.
• FreeStyle
™
MAX Reagent: A proprietary, animal origin-free formulation for
high transfection efficiency of plasmid DNA into CHO DG44 Cells (cGMP
banked). See page 12 for more information.
• CD OptiCHO
™
Medium: Defined, serum-free medium formulated for
selection and growth of CHO DG44 Cells (cGMP banked) expressing DHFR
and the recombinant protein of interest. See page 13 for more information.
• Additional components: OptiPRO
™
SFM (serum-free medium) for optimal
DNA:lipid complex formulation, L-glutamine for increased media stability,
Geneticin
®
Selective Antibiotic for stable cell line selection, and the surfactant
Pluronic
®
F-68 to control shear forces in suspension culture.
6