Nucleofector technology and transient protein production

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Nucleofector ® technology
and transient protein
production
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Nucleofector technology
The Nucleofector ® technology and
transient protein production
Transient protein expression systems have gained increasing
relevance as they enable fast and flexible protein production.
Protein amounts up to 10 mg can be produced within
a few days in contrast to production periods up to
several months using stably transfected cell clones.
Transient protein production offers:
› fast production of small scale protein amounts
› low costs for cell maintenance
Easy and fast nucleofection procedure:
harvest of
protein
Combine cells of interest, expression
vector and the appropriate celltype
specific Nucleofector ® Solution
and transfer to an amaxa certified
cuvette.
Insert the cuvette into the Nucleofector and choose the cell-type
specific Nucleofector program. Press the start button “X”. After a
few seconds nucleofection is completed. Rinse the cuvette with culture
medium and transfer the cells into the culture flask. For 1 x 10 8
cells, ten cuvettes have to be nucleofected.
Culture cells for several days.
In the past gene transfer into serum-free cultivated mammalian
cells suitable for protein production was hampered
by inefficient transfection methods.
The Nucleofector technology represents the ideal tool for
gene transfer even into hard-to-transfect cell lines like
suspension CHO cells.
It has now been successfully tested and validated for its use
in transient protein production.
Benefit from transient protein production using nucleofection:
› suitable for sCHO, sHEK-293, BHK-21, NS0 and many other
cells
› applicable even under serum-free conditions
› high transfection efficiencies
› low DNA amounts
page 2 www.amaxa.com Nucleofector technology

Nucleofector technology
Nucleofection ® is superior to lipofection
for transient protein production
In a comparative study the Nucleofector technology (amaxa)
and the lipofection reagent Lipofectamine 2000 (Invitrogen)
have been tested in regard to their suitability for transient
protein production.
Nucleofection achieves 10 -30 times higher expression rates
in CHO cells compared to cells transfected with lipofection. A
similar ratio has been determined for both the specific and
the volumetric productivities.
Experimental setup:
2 different suspension CHO cell clones have been transfected with a plasmid
encoding the human secreted alkaline phosphatase (SEAP) either using
Lipofectamine 2000 (Invitrogen) or nucleofection.
Transfection conditions:
Cell line
sCHO-clone1
(ECACC)
sCHO-clone2
(Invitrogen)
Transfection
method
Lipofectamine
2000
nucleofection
Lipofectamine
2000
nucleofection
Transfection
conditions
290 µl
Cell Line Kit V
Program U-024
580 µl
Cell Line Kit V
Program U-024
DNA
58 µg
pDRIVE-hSEAP
25 µg
pDRIVE-hSEAP
116 µg
pDRIVE-hSEAP
25 µg
pDRIVE-hSEAP
Cell number
5 x 10 7
(^= 5 samples)
5 x 10 7
(^= 5 samples)
5 x 10 7
(^= 5 samples)
5 x 10 7
(^= 5 samples)
Post transfection cells were cultivated as batches in spinner flasks containing
50 ml culture medium for 5 days under non-regulated conditions.
Suspension HEK-293 cells (DSMZ, adapted from adherent cell clone) have also
been tested successfully. 5 x 10 7 cells (^= 5 samples) have been nucleofected
with 25 µg pDrive-hSEAP using Cell Line Nucleofector Kit V in combination
with program X-001. A specific productivity of 2.4 µU/c/d and a volumetric productivity
of 10.5 U/ml could be obtained. These cells were not transfectable
using lipofection.
Specific productivities (µUnits/cell/
day) of hSEAP referred to a cell
density of 10 6 cells/ml seeded after
the respective transfection.
specific productivity hSEAP [µU/c/d]
10
9
8
7
6
5
4
3
2
1
0
lipofection
nucleofection
sCHO-clone1
sCHO-clone2
page 3 www.amaxa.com Nucleofector technology

Nucleofector technology
Volumetric productivities (Units/ml)
of hSEAP.
volumetric productivity hSEAP [U/ml]
45
40
35
30
25
20
15
10
5
0
sCHO-clone1, lipofection
sCHO-clone1, nucleofection
sCHO-clone2, lipofection
sCHO-clone2, nucleofection
time [d] 1 2 3 4 5
Efficient antibody production using
nucleofection ®
The Nucleofector technology was also tested successfully for
its suitability to produce functional proteins.
1.7 mg of a human IgG1 antibody was produced within five
days.
antibody concentration [µg/ml]
45
40
35
30
25
20
15
10
5
0
time [d] 1 2 3 4 5
Antibody production after nucleofection of CHO suspension cells
1 x 10 8 sCHO cells (clone 2, ^= 10 samples) were nucleofected using Cell Line
Nucleofector Kit V in combination with program U-24 or U-024. Cells were cotransfected
with plasmids encoding either the DNA sequence of the light chain
or the heavy chain of a human IgG1 antibody. Post nucleofection cells were cultivated
for 5 days in 50 ml serum-free medium. Cells reached a specific productivity
of 5.5 pg/c/d and a volumetric productivity of 39 µg/ml. Within 4-5 days
1.7 mg of the antibody could be produced (determined by ELISA). The integrity
of the produced antibody was verified via Western blotting.
q
For more scientific data go to www.amaxa.com/proteinproduction.html or contact our Scientific Support Team.
page 4 www.amaxa.com Nucleofector technology

Nucleofector technology
High transfection efficiencies in hard-to-transfect cells
such as sCHO and sHEK-293
CHO cells are the predominant cell line used for protein production. Suspension CHO cells
have so far been difficult to transfect by conventional non-viral methods. The same is true for
many other suspension cell lines used for protein production like BHK-21 and NS0.
High transfection efficiencies
after nucleofection.
Transfection efficiencies for two different
suspension CHO cell clones
and two different suspension HEK-
293 cell clones after nucleofection
of pmaxGFP. maxGFP positive cells
were determined 24h post nucleofection
by FACS analysis (light bars).
Viabilities were assessed after propidium
iodide staining by FACS analysis
(dark bars).
transfection efficiency/viability (%)
100
90
80
70
60
50
40
30
20
10
0
efficiency
viability
sCHO-clone1
sCHO-clone2
sHEK-293-clone1
sHEK-293-clone2
How to find your cell line of interest
The Nucleofector technology is the ideal tool for efficient non-viral gene transfer into different
suspension CHO and HEK-293 cell clones. Please find the Optimized Protocols for
these and many other cell lines on amaxa’s homepage.
Visit the amaxa online
cell line database
Enter your cell line
of interest or view
complete list
Your cell line of interest
Screen results
Cell type listed
Cell type not listed
Click on cell name
Use recommended Nucleofector
® Kit in combination with
the respective Optimized
Protocol or use the settings
optimized by other users.
Use Cell Line Optimization
Nucleofector ® Kit or contact
amaxa’s Scientific Support
Team for further advice.
page 5 www.amaxa.com Nucleofector technology

Nucleofector technology
How Nucleofector ® technology works
The Nucleofector ® Device
delivers unique electrical parameters that are different
from any commercially available electroporation device.
The electrical settings are pre-programmed and each program
is optimized for the requirements of a particular cell
type and can be used for the delivery of different substrates
such as siRNA duplexes, DNA vectors or RNA.
The Nucleofector ® Kits
contain amaxa specified cuvettes, pipettes, Nucleofector
Solution, Supplement and the positive control vector
pmaxGFP. For cell lines, three different Nucleofector Solutions,
R, T and V, are available. All solutions provide a cellfriendly
environment that ensure the highest transfection
results and cell viability. Nucleofector Kits are only functional
in combination with the Nucleofector ® Device.
Ordering information
Nucleofector ® Kits for cell lines*
VCO-1001
VCA-1001
VCA-1002
VCA-1003
Cell Line Optimization Nucleofector ® Kit
Cell Line Kit R
Cell Line Kit T
Cell Line Kit V
d
For an up-to-date overview or more detailed information about amaxa products, please see
www.amaxa.com/productlist.
* Each Kit contains Nucleofector ® Solution, Supplement, Cuvettes and Pipettes for 25 reactions and the pmaxGFP plasmid as a positive control.
VCO-1001 contains Nucleofector Solution ® , Supplement, Cuvettes and Pipettes for 50 reactions and the pmaxGFP plasmid as a positive control.
WKB-1020_05.2005
amaxa’s Nucleofector ® Process, Nucleofector ® Device and Nucleofector ® Solutions are covered by PCT Applications PCT/EP01/07348,
PCT/DE02/01489, PCT/DE02/01483, and other pending patents, and domestic or foreign applications corresponding thereto.
amaxa, Nucleofector, nucleofection and maxGFP are trademarks of amaxa GmbH.
Lipofectamine 2000 is a trademark of Invitrogen.
amaxa GmbH, Europe/World
amaxa Inc., USA
Scientific Support
Scientific Support
+49 (0)221-99199-400 (240) 632-9110
scientific-support@amaxa.com www.amaxa.com scientific-support.US@amaxa.com