User guide

Transfecting CHO DG44 Cells with FreeStyle
™
MAX Reagent for
single-subunit protein expression
Introduction
You will use FreeStyle
™
MAX Reagent to transfect suspension CHO DG44 Cells
(cGMP banked) with the pOptiVEC
™
expression construct containing your gene
of interest
IMPORTANT!
If you are expressing a single-subunit protein, you must generate your expression
construct using the pOptiVEC
™
-TOPO
®
TA vector. The pOptiVEC
™
expression
construct allows genomic amplification by MTX amplification and clonal selection
by limiting dilution in cloning medium as recommended on page
57.
Do not use an expression construct generated with the pcDNA
™
3.3 TOPO
®
TA
vector, because the CD-OptiCHO
™
Medium is incompatible with pcDNA
™
3.3
expression constructs.
Plasmid
preparation
The pOptiVEC
™
plasmid construct must be clean, sterile, and free from
contamination with phenol and sodium chloride for transfection into CHO DG44
Cells (cGMP banked). Contaminants may kill the cells, and salt interferes with lipid
complexing, decreasing transfection efficiency. We recommend isolating plasmid
DNA using an endotoxin-free or a low-endotoxin kit such as the PureLink
®
HiPure
Plasmid Midiprep DNA Kit (see page 71 for ordering information).
Linearizing the
plasmids
Prior to using the Freedom
™
DG44 Kit to transfect CHO DG44 Cells (cGMP
banked) with your pOptiVEC
™
construct containing your gene of interest, you
may linearize the plasmid. Linearizing your vector may not improve transfection
efficiency, but it increases the chance that the vector will integrate into the host
cell genome without disrupting the gene of interest or other elements required for
expression in mammalian cells.
• We suggest using Pvu I, which cuts once in the ampicillin resistance gene on
the plasmid. Other unique restriction sites are possible. Complete restriction
map of pOptiVEC
™
-TOPO
®
TA is available at www.lifetechnologies.com. Be
sure that your insert does not contain the restriction enzyme site you use to
linearize the vector.
Note: If an appropriate linearization site is not present, you may transfect the circular
plasmid. Transfection efficiency will not be affected.
• After digestion, precipitate the DNA, resuspend pellet in sterile water, and
re-quantify using your method of choice.
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