› amaxa news # 5
amaxa news
#5
New
›
Nucleofection® of mouse T cells
and human monocytes
›
amaxa´s mission 2005
›
Nucleofector® technology and
stable expression
›
Broad-range transfection Basic Nucleofector® Kits
gene transfer begins here
Editorial
In 2005 amaxa is aiming high. Our mission is to complement our
selection of transfection protocols and kits to address the 100 most
requested cell lines and primary cells.
Light up
your cells!
Among primary cells, one of those “most wanted cells” has
been beyond any doubt the mouse T cell. With amaxa’s newest
With amaxa´s expression vectors.
developments this cell type can now be ticked off from the “most
wanted” list.
Additionally, we have recently expanded our immunology portfolio
by introducing the Human Monocyte Nucleofector ® Kit. With
the launch of these two new kits we are taking the opportunity
to focus our activities this spring on the immunologists’ needs.
Get more detailed information on both the Mouse T Cell and
Human Monocyte Nucleofector® Kits and how you can benefit from
working with them by reading pages 4-7.
new
pmaxFP-Green,
pmaxFP-Red
and pmaxFP-Yellow
> extremely bright fluorescence
> promoterless vectors and vectors for
N-, C-terminal fusions available
> affordable
> no license fee for industry during first
6 months*
In the field of transient gene transfer there is no better technology
for primary cells and hard-to-transfect cell lines than amaxa´s
Nucleofector technology. But, did you know that it is also highly
suitable for stable transfections? This issue of ‘amaxa news’ holds
valuable information on sustained gene expression (pages 9-12).
Finally we allow a glimpse into our labs. Scientists have built up
amaxa and are still at the heart of the company. Take a look behind
the benches and get a closer impression of our R&D department.
Call us now for more details.
Of course there is a lot more to discover. Have fun reading
* applicable for R&D purposes only
amaxa Europe / Export
[email protected]
+49 (0) 221 99199-400
amaxa USA
[email protected]
240-632-9110
‘amaxa news #5’!
Rainer Christine
CEO
› Table of contents
New products
4
Nucleofection® of mouse T cells and human monocytes
Hot topic
8
Nucleofected mouse T cells respond to cytokine gradient
Teresa W. Wu, Thomas O. Cameron and Michael L. Dustin
Hot topic
9
Nucleofector® technology and stable expression
Technote
10
Use of Nucleofector® technology to establish stably
expressing cell lines
Hanns-Martin Schmidt, Markus Zumbansen, Rainer Wittig, Stephanie Blaich,
Lisha Brown, Stefan Lyer, Annemarie Poustka, Jan Mollenhauer and Michael Nix
New products
13
amaxa´s mission 2005: Transfect the 100 most wanted cell types
Hot topic
14
Making the most of your RNAi experiments
New products
16
Basic Nucleofector® Kits – check new perspectives for your
research with primary mammalian cells
Application note
17
Mouse ES cells – a promising tool in molecular genetics
Hot topic
18
Nucleofection® of stimulated mouse T cells
amaxa worldwide
19
amaxa Down Under – “G´day” to our Australian distributor
Integrated Sciences
amaxa insights
20
R&D: Journey to the heart of amaxa
FAQ
22
Frequently asked questions on Nucleofector® technology in
immunology research
amaxa insights
[k]
23
Ordering information
To order products contact our
Scientific Support Team (see back
cover for contact details).
q
Supplementary information
Editor
Wolfgang Kintzel
Editorial board
Bernd Eschweiler, Katrin Höck,
Andrea Toell
Production coordinator
Benno Limberg
Please note that to date amaxa’s
Nucleofector® technology is intended for
research use or investigational use only.
Design
vierviertel – Agentur für
Kommunikationsdesign GmbH
[email protected]
To order supplementary information
complete the reply card in the center
of this newsletter.
amaxa web information
To instantly find more detailed
information on certain subjects visit
the indicated website.
› page 3
Meet amaxa in 2005
amaxa news is published by
amaxa GmbH
Nattermannallee 1
50829 Koeln
Germany
[email protected]
Tel: +49(0)221-99199-0
Fax:+49(0)221-99199-1 1 1
› www.amaxa.com
© March/2005 amaxa.
All rights reserved. Printed in Germany.
› New products
new
Nucleofection® of mouse T cells
and human monocytes
- introducing a new approach to achieve this breakthrough
Has the transfection of primary mouse T cells and human monocytes been a major obstacle for
your research? Nucleofector® technology already enables transfection of a wide range of primary
cells relevant to immunology research. Now we have achieved a further breakthrough by introducing the Nucleofector Kits for mouse T cells and human monocytes. It turned out that achieving efficient nucleofection results required a new approach. This involved the well known components of the Nucleofector technology - optimized electrical settings and Nucleofector solution,
plus specialized culture media for post-nucleofection recovery of transfected cells. Take a closer
look and get to know the new Nucleofector Kits and the impact they can have on your immunology research.
Immunology/hematology will remain a center focus of amaxa’s research activities in 2005.
Click on www.amaxa.com/immunology to be informed when our future products - Nucleofector
Kits for human and mouse macrophages - will be available.
› page 4
› amaxa news # 5
› New products
new
Mouse T cell nucleofection®
- reliable transfection for C57BL/6 and BALB/c
Efficient transfection of mouse T cells has to date only been possible through virus-based protocols,
and even these methods were effective only on activated T cells. amaxa’s Nucleofector® technology
has changed all that, making it possible to transfect unstimulated mouse T cells from C57BL/6 and
BALB/c mice without the use of viruses. With this new tool in hand you can now conduct detailed studies on mouse T cell function, activation, regulation and signaling that have been impossible so far.
With the Nucleofector technology a non-viral alternative is now available:
First non viral transfection of mouse T cells
› Ability to study important events in T cell activation
Reliable performance
› Up to 40% efficiency
› Transfected T cells can be stimulated post nucleofection
› Evaluated for the most commonly used mouse strains:
and differentiation
Functionality
Relevant for your research
C57BL/6 and BALB/c
› Optimized Mouse T Cell Nucleofector® Medium included
Complete solution
The Mouse T Cell Nucleofector® Kit has been evaluated for CD4+
and CD8+ T cells from C57BL/6 and BALB/c mice. Figure 1 gives
C57BL/6
% transfected cells
ciencies at 24 h are 23% (C57BL/6) and 39% (BALB/C) (data not
shown). The viabilities post nucleofection for CD4+ cells are betto 41% at 24 hours post nucleofection can be achieved (data not
shown).
48h
60
range between 27 and 37%. For CD8+ T cells transfection effi-
ween 25% and 55% (Figure 2). For CD8+ cells viabilities of 35%
24h
70
an overview of transfection efficiencies for CD4 + T cells from
both strains. Average transfection efficiencies for CD4+ cells
BALB/c
50
40
30
20
10
0
CD4+
CD4+
Fig. 1: Average transfection efficiencies for CD4+ mouse T
cell from C57BL/6 and BALB/c strains. Cells were transfected with pmaxGFP™ and analyzed at 24 and 48 hours post
nucleofection by flow cytometry.
Optimized Mouse T Cell Nucleofector® Medium –
C57BL/6
BALB/c
®
% living cells (PI negative)
a new component of the Nucleofector technology
Unstimulated primary mouse T cells in culture show a high
mortality with significant cell loss. In untransfected control samples already at 24 hours after isolation 35-40% (C57BL/6) and
25-30% (BALB/c) PI positive cells can be observed. Due to this
reason, we have developed a post-nucleofection recovery
medium, in addition to all the other well known components
› page 5
48h
50
40
30
20
10
0
CD4+
medium that helps reducing cell loss. Therefore, the Mouse T
Cell Nucleofector® Kit contains 100 ml of a specialized culture
24h
70
60
CD4+
Fig. 2: Average viabilities (% PI negative) for CD4+ mouse T
cells. Mouse T cells were transfected with pmaxGFP™ and analyzed by flow cytometry at 24 and 48 hours after nucleofection.
› www.amaxa.com
› New products
of the Nucleofector Kits. The medium is used for the culture
RPMI 1640
of nucleofected T cells and it supports survival of nucleofected
enhancement of nucleofected T cells cultured in Mouse T Cell
Nucleofector ® Medium vs. those cultured in RPMI medium. An
increased viability of 10% to 20% can be observed at 24 hours
post nucleofection. You can use the Mouse T Cell Nucleofector®
% living cells (PI negative)
T cells post transfection. Figure 3 demonstrates the viability
Nucleofector
Medium
60
50
40
30
20
10
0
CD4+
Medium with any of your antibiotics or cytokines regularly used
CD8+
for your mouse T cell culture.
What’s on your mind? – Go for new challenges in your
mouse T cell research
Are you interested in mouse T cell activation or signaling? Do
Fig. 3: Mouse T Cell Nucleofector® Medium increases viability post nucleofection. Primary C57BL/6 mouse T cells were
transfected with pmaxGFP™ and cultured in RPMI 1640 or
Mouse T Cell Nucleofector® Medium both supplemented with
5% FCS. 24 hours post nucleofection viability (%PI negative)
for CD4+ and CD8+ cells was determined by flow cytometry.
you wish to perform functional assays or stimulation experiments on transfected mouse T cells? Just go ahead and
remained unanswered forever.
Many of these assays in mouse T cell research need to be done
in stimulated T cells. Of course, nucleofected T cells can be
activated with stimuli, such as aCD3/aCD28 antibodies, to
study the function of your transgene in stimulated T cells.
Mouse T cells nucleofected with pmaxGFP™ and subsequently
stimulated show expression of activation markers, such as
% CD25 expressing CD8+ cells
address some of your scientific questions that seem to have
48h
100
72h
80
60
40
20
0
-Nucleofection/-DNA
+Nucleofection/-DNA
+Nucleofection/+DNA
CD25, that is comparable to control samples (Figure 4).
The response of T cells to certain chemokines is commonly
determined by transmigration assays. This is of particular interest in T cells transfected with transgenes mediating chemokine receptor function. The proper behavior of pmaxGFP™
nucleofected T cells in a transmigration assays is demonstrated
on page 8 of this newsletter by Michael Dustin and co-workers.
Fig. 4: Mouse T cells can be stimulated after nucleofection.
Primary C57BL/6 mouse T cells were transfected with 2.5 µg
pmaxGFP™. 3 h post nucleofection cells were stimulated with
plate-bound anti-CD3e (5 µg/ml) and anti-CD28 (2 µg/ml)
antibodies. 48 and 72 h post nucleofection, CD8+ cells were
analyzed for CD25 surface expression. Figure shows proportion
of CD25 expressing living CD8+ T cells. (%CD25 expression in
unstimulated samples ranged from 10-20%).
They have used this assay in combination with nucleofection as
a major tool to study chemokine receptor function.
Even more is possible with the Mouse T Cell Nucleofector® Kit!
Please take a look at page 18 to view an example for transfection
of mouse T cells stimulated prior to nucleofection.
[k]
Ordering information
Mouse T Cell Nucleofector® Kit
q
Cat. No.: VPA-1006
amaxa web information
www.amaxa.com/immunology
› page 6
› amaxa news # 5
Supplementary information
Immunology Flyer
› New products
new
Human monocyte nucleofection®
The Human Monocyte Nucleofector® Kit introduces the first non-viral transfection method for primary
monocytes. With the new combination of Nucleofector Kit plus specialized Nucleofector Medium
transfection efficiencies of up to 70% can be achieved. The Kit further extends the line of Nucleofector Kits for primary cells of the hematopoietic cascade. Go to www.amaxa.com/immunolgy to
view a complete list of cells relevant to your research that can be transfected with Nucleofector
technology. Watch for news on our next targets: nucleofection of mouse and human macrophages!
48h
90
80
80
70
cytes you can now address topics such as:
60
50
% transfected cells
cellular immunology
signaling
differentiation and activation
100
90
% living cells (PI-negative)
With the efficient transfection of human mono-
›
›
›
›
›
24h
100
Choose your topic
40
30
20
10
0
inflammation
Transfection efficiency
24h
48h
70
60
50
40
30
20
10
0
Viability
Fig. 1: Average transfection efficiencies and viabilities (%PI
negative) of human monocytes transfected with pmaxGFP™
and cultured in Human Monocyte Nucleofector® Medium.
and many more
Human monocyte nucleofection at a glance
7x10 5
With nucleofection you can achieve transfection efficiencies up to
6x10 5
5x10 5
previously impossible. Figure 1 summarizes transfection efficiency
4x10 5
and viability of monocytes.
Just like the Mouse T Cell Nucleofector® Kit, the Human Monocyte
no. of living cells
70% allowing you to perform many functional analyses that were
3x10 5
2x10 5
RPMI
1x10 5
0x10
Nucleofector® Kit contains 100 ml of a specialized recovery
Nucleofector
Medium
5
PBMC preparation
medium that is required to improve post-transfection survival of
nucleofected monocytes. As shown in Figure 2 for a representative PBMC preparation, the Human Monocyte Nucleofector®
Medium elevates the number of viable cells post nucleofection as
compared to culture in regular RPMI medium.
[k]
Fig. 2: Human Monocyte Nucleofector® Medium increases
number of living monocytes post nucleofection. Primary
human monocytes were nucleofected with pmaxGFP™ and
cultured in RPMI 1640 or Human Monocyte Nucleofector®
Medium. 24 hours post nucleofection absolute numbers of
viable monocytes were determined by flow cytometry.
Ordering information
Human Monocyte Nucleofector® Kit
q
Cat. No.: VPA-1007
amaxa web information
www.amaxa.com/immunology
› page 7
› www.amaxa.com
Supplementary information
Immunology Flyer
› Hot topic
Nucleofected mouse T cells
respond to cytokine gradient
Teresa W. Wu, Thomas O. Cameron, and Michael L. Dustin, New York University School of Medicine,
Skirball Institute of Molecular Medicine, Division of Molecular Pathogenesis, New York, NY, USA.
Lymphocyte trafficking is largely determined by
10 million splenocytes freshly isolated from BALB/c mice were
the chemotaxis of cells bearing particular che-
nucleofected with 2 µg pmaxGFPTM plasmid following the Opti-
mokine receptors towards regions containing
mized Protocol for murine T cells (amaxa). 24 h post nucleofec-
their cognate ligands (chemokines). The ability
of cells to respond to particular chemokines
tion, cells were analyzed for maxGFPTM expression. About 20% of
the CD4+ T cells were maxGFP-positive, as determined by FACS
is commonly tested in vitro by transmigration
analysis. Compared to cells incubated overnight at 4°C, about
assays in which cells crawl through a membrane
75% live cells were found 24 h after transfection (Fig. 1).
with 1-5 micron-sized pores towards a chemokine-rich media. Naïve T cells express the che-
The percentage of pmaxGFP-positive cells is comparable between
mokine receptor CCR7 and therefore respond
input cell population and the transmigrated population, no signi-
robustly to the cognate ligand SLC (CCL21). We
ficant difference is observed (Fig. 2A). Non-nucleofected and
examined whether Nucleofection disturbs this
nucleofected BALB/c lymphocytes transmigrated in response to
normal behavior of T cells.
SLC with nearly identical efficiency (Fig. 2B). Together, these data
suggest that nucleofection of murine T lymphocytes will be an
invaluable tool for the study of chemokine receptor function.
A
9
B
Cell population
25
60
8
5
3
2
% Transmigrated
% GFP-positive
Live cells (Millions)
6
4
15
10
5
1
0
0
4°C overnight
pmaxGFP
no SLC
+SLC
50
20
7
Cell population
40
30
20
10
0
Input
Transmigrated
4°C overnight
pMaxGFP
Fig. 1: Murine T cells were either stored at 4°C after isolation or transfected with pmaxGFPTM plasmid. The number
of live cells was determined after 24 h by propidium iodide
exclusion. In the example shown here 75% of T cells survived
the treatment.
Fig. 2: Analysis of nucleofected murine T cells in a transmigration assay
towards SLC. (A) The percentage of maxGFPTM-positive cells is nearly identical in
the input population and the transmigrated population. (B) maxGFP TM-positive
cells display same migration activity as untreated cells. These data suggest that
transfected cells do not show impaired migration behavior.
› page 8
› amaxa news # 5
› Hot topic
Nucleofector® technology and
stable expression
The Nucleofector technology is well established as an efficient method to achieve transient transgene expression in
cell lines and primary cells. Its potential has also been demonstrated by applications such as siRNA-mediated gene
knock-down. Stable expression of genes is also an important application relevant for many research topics. Read on
and learn how to use the Nucleofector technology to generate stably expressing cell lines. For detailed information
please refer to the technote on pages 10 - 12 of this amaxa news.
Stable gene expression is used for various research applications, such as protein production or generation of cell lines for long-term functional analysis. Nevertheless, stable gene expression is
frequently hindered by a low number of integration events after gene transfer, often due to inefficient transfection. This is of special importance for hard-to-transfect cell lines which, transfected
with standard methods, normally show very low efficiencies. Nucleofector technology, which usually
achieves higher transfection efficiencies than conventional transfection methods, is thus well suited
to generate stably expressing clones from various cell lines, including those hard-to-transfect.
The technote on the following pages demonstrates the use of nucleofection for different aspects of
stable transfection such as:
›
›
›
›
stable expression of proteins in standard cell lines and hard-to-transfect cell lines
stable expression in suspension and adherent cell lines
generation of single-integration clones
batch culture and single clone approaches
How to use the Nucleofector technology for stable transfection
Do you aim at using the Nucleofector technology for stable transfection of a hard-to-transfect cell
line to generate a single-integration clone? Or do you have other goals in mind involving stable
transfection? One parameter you don’t have to worry about is the nucleofection conditions to
achieve stable expression. Simply follow the transfection conditions described in the Optimized
Protocol. Certain other parameters, however, such as DNA amounts and properties, selection or
culture conditions, have to be adapted according to your needs.
=
Nucleofection conditions
as described in the Optimized Protocol
✓
adapt to your needs
!
DNA properties (linear - circular)
DNA amount
Culture conditions
Selection conditions
› page 9
› www.amaxa.com
› Technote
Use of Nucleofector® technology
to establish stably expressing
cell lines
Hanns-Martin Schmidt 1*, Markus Zumbansen 1*, Rainer Wittig 2, Stephanie Blaich 2, Lisha Brown 3,
Stefan Lyer 2, Annemarie Poustka 2, Jan Mollenhauer 2, and Michael Nix 1
The number of stably expressing clones resulting from standard transfection experiments is often low, especially for
cells that are difficult to transfect. This limits many research applications. We show here that, besides being a very efficient method for transient expression of genes, the Nucleofector® technology can also be used to easily generate stably
expressing batch cultures or clones with considerable efficiency. Although a general protocol cannot be given, the data
shown here may serve as a guideline for fast establishment of stable transfection protocols for various cell lines.
1
amaxa GmbH,
Köln, Germany;
2
Department of
Molecular Genome
Analysis, German
Cancer Research
Center (DKFZ),
Heidelberg,
Germany;
3
Department of
Urology, University of Washington, Seattle, WA,
USA. *These
authors contributed equally.
Introduction
Stable expression of genes is important
for diverse fields of molecular and cellular
biology. Objectives range from the quantitative production of proteins to the stable
correction of genetic defects. In cases
where non-homologous recombination
events suffice to achieve the desired
effect, experiments are often limited by
either a low number of stably expressing
cells, mostly due to inefficient transfection or by the inherent disadvantages of
viral systems. We show here, that stably
expressing cells are established from
a standard cell line (CHO-K1) as well as
from several hard-to-transfect suspension
(K562) or adherent cell lines (e.g. A549,
C4-2) by using the Nucleofector® technology. Different culture conditions after
transfection have been analyzed. In
some application fields it is necessary to
generate clearly characterized cells derived from a single transfected clone. This
may be achieved by the limiting dilution
procedure as outlined here with CHO cells.
In other cases a thorough definition of
clones may not be required, e.g. for over-
expression of a secreted protein as shown
here with bone morphogenic protein-7
(BMP-7). These cells can then be cultured
in one single batch.
Gene transfer of plasmid DNA via transfection often results in multiple integration events. This may be a consequence
of the entry of multiple plasmids, or of amplification and/or concatamerization of
plasmid DNA once it is inside the cell. The
ability to produce clonal cell lines derived
from a single integration event can be an
advantage as it simplifies genetic and
functional analyses, minimizes gene
dosage effects and reduces the possibility
of integration-dependent disruption of
genetic pathways. Hence, we evaluated
the ability to generate single-integration
clones in several cancer cell lines using
Nucleofector technology.
Results
Stable transfection of CHO-K1 cells
CHO-K1 cells (ATCC Cat. No. CCL-61, passage 8–21) were nucleofected with different amounts of circular and linearized
pmaxFPTM- Green-C plasmid (amaxa) con-
A
transient
stable
# of wells/plate
with living cells
CHO-K1
transfection
efficiency
24h p.N.
circular plasmid
linearized plasmid
taining a neo cassette and the Pontellina
plumata GFP protein under a CMV promoter following the instructions in the Optimized Protocol (amaxa). Cells were grown
without selection for 24 h, counted by flow
cytometry and plated onto 96-well plates
at a mean concentration of 10 cells per
well. They were further grown under the
selection pressure of 700 µg/ml G418.
After every 7-10 days, 100 µl of fresh
medium with G418 was added per well.
CHO-K1 cells were analyzed after 3-4
weeks under selection pressure using a
96-well plate reader (Tecan). Living cells
were identified by a WST-1 assay (Roche)
with a spectrometric absorption readout
at 450 nm and a reference wavelength of
600 nm. GFP expression was characterized by fluorescence measurement. The
respective thresholds were chosen, such
that they were in good agreement with
random sample visual checks done by
light and fluorescence microscopy.
Transient transfection efficiencies were
high with both linear and circular DNA.
The results summarized in Table 1 A
demonstrate that increasing the amount
# of wells/plate
% of wells/plate with
with GFP-expressing stably expressing
and living cells
living GFP cells
2 µg
94 %
+/-
2%
20
+/-
4
12
+/-
5 µg
97 %
+/-
1%
69
+/-
11
48
+/- 14
50 %
2 µg
31 %
+/-
4%
46
+/- 10
24
+/-
8
25 %
5 µg
65 %
+/-
3%
72
+/-
51
+/-
6
53 %
8
2
13 %
Table 1 A: Higher DNA amounts lead to more wells with stably expressing CHO-K1 cells. Data are results of the analyses of
3 different experiments with 2 µg DNA and 2 experiments with 5 µg DNA. Results are shown from 6-9 transient transfections
each. Cells were seeded at a mean density of 10 cells/well and cultured for 3-4 weeks under selection pressure. The resulting
number of living or GFP-positive cells, respectively, refer to wells per 96-well plate.
› page 10
› amaxa news # 5
› Technote
B
transient
stable
# of wells/plate
with living cells
K562
transfection
efficiency
24h p.N.
# of wells/plate
% of wells/plate with
with GFP-expressing stably expressing
and living cells
living GFP cells
circular plasmid
2 µg
82 %
+/-
5%
36
+/-
7
25
+/-
6
26 %
linearized plasmid
2 µg
30 %
+/-
4%
43
+/- 15
30
+/- 13
31 %
Table 1 B: In K562, cicular and linear plasmids yield comparable numbers of wells with stably GFP expressing cells. Data
are results of the analyses of 4 different cell batches. Transient transfection efficiencies were determined from 12 samples. After
one week under selection, cells were seeded at a mean density of 100 cells/well and cultured for 4 weeks under selection. The
resulting number of living or GFP-positive cells, respectively, refer to wells per 96-well plate.
of DNA leads to a higher number of stably
expressing clones. About 50% of wells
contained GFP-expressing cells when
5 µg linear or circular DNA had been used.
With 2 µg of DNA, linearized plasmid
led to twice as many clones as circular
DNA (25% vs. 13% of wells contained
expressing cells).
Stable transfection of K562 cells
Suspension cells are difficult to transfect
with conventional methods, e.g. lipidbased reagents, which makes it difficult
per se to obtain a satisfying number of
stably expressing cells. We transfected
K562 cells (ATCC, Cat. No. CLL-243,
passage 10-13) with 2 µg of linearized or
circular plasmid following the respective
Optimized Protocol (amaxa). Cells were
grown without selection for 24 h, counted
by flow cytometry and plated onto 6-well
plates at a cell density of 2x105 living
cells/ml. Cells were continuously grown in
6-well plates in medium containing 500
µg/ml G418. Growth behavior and efficacy
of the selection was followed by flow
cytometry, analyzing the ratio of living
and dead cells. After one week cells were
Medium
plated onto 96-well plates at a mean
density of 100 cells per well. Analysis was
done after 4 weeks of G418 addition.
Since the culture medium for K562 cells
cannot be changed without considerable
cell loss we chose to grow cells without
change of media, i.e. tolerating pH-related change of media color. These wells
were counted and analyzed on a fluorescence microscope for GFP expression.
Table 1 B shows the results of the analyses of four different cell batches with
at least three independent samples each.
Transfection efficiencies of about 30%
with linearized plasmid and about 80%
with circular plasmid were observed
24 h after nucleofection with mortalities
being low with both isomers. After selection, 30-40% of the wells contained
living cells, about 70% of which also
expressed GFP.
Protein production in prostate cancer
cell lines
Bone morphogenetic proteins (BMPs)
regulate many developmental processes
and are involved in bone formation.
BMP-7 has been reported to be up-regu-
Cell lysate
(2
-1)
Nucleofector®
Program
Approximate
Efficiency
U87MG
V
A-23
50%
U138MG
R
T-01
70%
A549
T
A-23
40%
pc
DN
A
Fig. 1: Efficient protein production in
hard-to-transfect cancer cell lines.
Western blot analysis of BMP-7 production by stably transfected C4-2 cells. The
first clones (1-2) produce BMP-7 at 5.4
ng/ml/106 cells, the other clones (2-1)
produce BMP-7 at 8.5 ng/ml/106 cells as
analyzed by anti-BMP-7 staining (R&D
Systems). Protein was neither found in
cells transfected with empty pcDNA
vector nor in the cell lysates.
› page 11
Nucleofector®
Solution
C4
-2
(12)
BM
P7
BM
P7
C4
-2
pc
DN
A
C4
-2
(2
-1)
C4
-2
BM
P7
C4
-2
C4
-2
BM
P7
(12)
Cell Line
lated in prostate cancer bone metastases.
Empty pcDNA3.1 vector (Invitrogen) or
pcDNA3.1 containing cDNAs coding for
bone morphogenic protein-7 (BMP-7)
were linearized.
1 µg of linearized vector DNA was mixed
with one million each of PC-3, C4-2,
or C4-2B cells in 100 µl Nucleofector
Solution V (amaxa) and subjected to
nucleofection. Cells were plated in 6-well
plates and cultured in RPMI supplemented with 10% FBS. Low cell death rates
were observed. 18 h post transfection the
medium was replaced by fresh medium.
After 3-5 days when cells began to proliferate, selection was started using media
containing 600 µg/ml G418. Stable batch
cultures were seen after 10 days. The stably expressing cells were maintained in
media containing 300 µg/ml G418. The
total population of C4-2 cells was assayed
by enzyme immunoassay (BMP-7 EIA,
R&D Systems) for the production of the
protein of interest and found to produce
up to 8.5 ng per ml medium per one million cells (Figure 1). Similar results were
obtained with the other cell lines.
Table 2: Transient transfection efficiencies of various cancer cell lines. Cells
were nucleofected with 5 µg circular plasmid DNA and analyzed for GFP expression
24 h post nucleofection. Efficiencies were approximately 50% lower when linearized
DNA was used.
› www.amaxa.com
› Technote
A
B
A549
M
1 2* 3**
U87MG
4 5* 6**
U138MG
M
1 2** 3** 4** 5** 6** 7**
* linearized plasmid
** circular plasmid
Fig. 2: Single integration events are more likely to happen with circular plasmids
than linearized. Southern blot analysis of transgenic clones created by nucleofection of tumor cell lines. (A) A549 were either nucleofected with linearized plasmid
(lane 2) or circular plasmid (lane 3). U87MG cells were nucleofected with linearized
(lane 5) or circular plasmid (lane 6). Lanes 1 and 4 show non-nucleofected controls of
A549 and U87MG cells respectively. (B) U138MG cells nucleofected with circular
plasmid showed single integration (lanes 3, 4, 5 and 7), double integration (lane 2)
and triple integration (lane 6). Lane 1 shows a non-nucleofected control. M = marker.
Generation of clonal single-integration
transgenic cell lines
To find out which nucleofection conditions are most appropriate to get
single random integration events we
tested three different cell lines: U87MG
and U138MG, derived from two human
glioblastomas; and A549, derived from
a human lung cancer. The vector used
for transfection contained an eGFP
reporter gene for the detection of
transfection efficiency as well as a neomycin phosphotransferase gene to enable selection with G418. 2x106 cells of
each cell line were nucleofected with
5 µg of vector (purified using Qiagen ®
EndoFree® Maxi-Kit, either linearized or
circular), using Nucleofector Solutions
and programs as indicated in Table 2.
Following nucleofection, cells were seeded into 6-well plates. The efficiency
of nucleofection was assessed by visual inspection after 24 h, using a Zeiss
Axiovert 25 fluorescence microscope.
All cell lines were efficiently transfected
as indicated in Table 2. After 24 h, cells
were trypsinized and re-seeded at 2x105
cells/ml into 10 cm cell culture dishes in
the presence of G418 (G418 concentrations: U87MG, 700 µg/ml; U138MG,
200 µg/ml; A549, 1 mg/ml). After 2-3
weeks, cell populations were again
inspected using fluorescence microscopy for the presence of eGFP-positive
colonies.
U87MG, and A549 cells formed distinct
colonies, with varying numbers of eGFP-
› page 12
negative cells. Colonies were picked and
seeded into 24-well plates. After reaching confluence cells were diluted and
re-plated into 96-well plates at a mean
concentration of 0.4 cells/well. U138MG
cells tended to spread across the entire 10 cm dish without forming distinct
colonies, with about 70-80% eGFPpositive cells. These cells were then
trypsinized, diluted, and re-plated into
96-well plates at a concentration of
0.4 cells/well.
After one week, the plates were visually
inspected for eGFP-positive colonies.
Positive colonies were expanded and
genomic DNA was isolated to characterize these clones. Single or multiple
integration of the transfected vector
into genomic DNA was assessed using
Southern blot analysis with a probe
specific for eGFP-ORF.
In U87MG and A549 cells, nucleofection using linearized plasmid
produced clones with multiple integration events, as shown by the binding
of eGFP-ORF probe to DNA fragments
of multiple sizes (from a single sample)
in a Southern blot analysis (Figure 2A).
However, nucleofection of A549 cells
using a circular plasmid produced
clones characterized by single-integration events (Figure 2A). Transfection of U138MG was only performed
with circular plasmid and produced
both single-integration and multipleintegration clones (Figure 2B).
› amaxa news # 5
Conclusions
We show here that the Nucleofector
technology can be equally used to
transfect multiple cell lines transiently
with high efficiencies and to generate
stably expressing batch cultures and
clones. Transient and stable transfections are shown for adherent and
suspension cell lines, including those
that are considered hard-to-transfect.
Stably transfected cells are generated
following standard nucleofection conditions using circular or linearized plasmid DNA. A higher DNA amount generally leads to higher clone numbers.
Linear DNA apparently results in
slightly higher rates of resistant cells
and most probably in higher integration
rates. This may be advantageous, if, for
instance, high protein expression rates
are desired, as shown here for BMP-7
expression. Circular DNA showed a
higher tendency to result in singleintegration events. However, from our
findings we conclude that integration
frequency seems to be influenced by
cell-type specific factors. We conclude
that it is impossible to give general
recommendation for the stable transfection of cells lines. Rather, for every
cell type of interest the ideal form of
plasmid DNA has to be established, just
like the culture conditions, seeding
densities, G418 concentrations as well
as method of clonal analysis.
› Hot topic
amaxa’s mission 2005:
Transfect the 100 most wanted
cell types
When amaxa was founded in 1998 the goal was to revolutionize the world of transfection by developing a non-viral transfection technology for primary cells. What resulted from this idea so far is the
Nucleofector technology which is now widely used in many laboratories around the world. Many formerly untransfectable cells, such as human and mouse T cells, have already been conquered by amaxa’s R&D team. amaxa’s mission for 2005 is now to fill the gaps by providing transfection solutions
for the remaining most wanted cell lines and primary cells.
The Nucleofector technology is the only nonviral transfection method that enables DNA to
directly enter the nucleus. Therefore, even nondividing cells, such as primary neurons or resting
blood cells, can be transfected with high efficiency. Furthermore, other substrates, such as siRNA
duplexes or mRNA, can also be efficiently delivered into hard-to-transfect cells to achieve
gene knock-down or transgene expression. And
Nuclefector™
II
Nuclefector™
II
start start
enter enter
exit exit
regardless of the substrate you transfect, it is
always done with the same cell type-specific
Nucleofector Kit and program.
In 2005, part of amaxa’s mission will be to con-
Apart from that, amaxa has dedicated itself to providing Opti-
tinue providing non-viral transfection solutions
mized Protocols for many more hard-to-transfect cell lines. To be
for primary cells, some of which have so far not
sure that your cell type is included in our development list, let us
been transfectable even by viral methods. With
know what your most wanted cell type is by filling out the reply
this mission in mind, kits currently under develop-
card in the center of our newsletter.
ment in amaxa’s R&D team include, among
others, those for macrophages (human and
mouse) and hepatocytes (multiple species).
q
To fulfill our mission in 2005, we have enlisted the help of two experts:
ElectroBoy and SolutionGirl. If you want to be updated regularly on their adventures and achievements, just fill out
the reply card in the center of the newsletter.
amaxa web information
www.amaxa.com/mission2005
› page 13
› www.amaxa.com
› Hot topic
Making the most of your
RNAi experiments
While RNA interference (RNAi) is a truly powerful and versatile mechanism, there are some important
considerations to keep in mind when designing your experiments using RNAi as a technique. Here, illustrated with examples from three publications out of the approximately 400 papers published by
amaxa users to date (www.amaxa.com/citations), we summarize some of these issues.
siRNA design - all siRNAs are not created equal
Van De Parre et al. [1] for example successfully
While delivery of the most potent siRNAs results in > 90% re-
used nucleofection of mRNA instead of DNA
duction in target RNA and protein levels, the effectiveness may
plasmid for expression in a human macrophage
vary between different siRNAs targeted to different regions of the
cell line.
same gene. An example for such variation is given in Fig. 1. Thus,
it is important to test several different siRNAs for each gene you
Non-specific effects (and appropriate controls)
wish to target.
Although keeping siRNAs < 30nt avoids activating the protein kinase PKR and 2’,5’-oligoade-
Duplexes or plasmid?
nylate synthetase pathways, siRNAs have still
As the down-regulation produced by siRNA duplexes is transient
been demonstrated to elicit non-specific effects,
in mammalian cells, several groups have constructed siRNA-
including both stimulation and repression of
expressing plasmid vectors in order to allow persistent siRNA
non-target genes [2].
expression. While these vectors offer some advantages over
siRNA duplexes, there are also some limitations.
B
A
SMYD3
you are designing new sequences it is prudent to first test their
b-actin
90
60
30
0
siR
considerably better than those for plasmids. For cells that are
SNU475 cells
120
M
NA oc
-E k
G
siR FP
N
siR A-1
N
siR A-4
N
siR A-12
siR N
NA A-1
4
siR -12
NA mm
-12
sc
r
2. Transfection efficiencies for siRNA duplexes (and mRNAs) are
siR
efficiency as siRNA duplexes prior to constructing new vectors.
M
NA oc
-E k
G
siR FP
N
siR A-1
N
siR A-4
N
siR A-12
siR N
NA A-1
siR -12 4
NA mm
-12
sc
r
time and labor-intensive than using duplexes. In particular, if
Relative absorbance
1. Generating vectors against new targets is considerably more
difficult to transfect (such as many primary cells), using duplexes may be your best means of observing an RNAi effect in
sufficient numbers of cells.
However, with nucleofection, many cells which are difficult to
transfect by other means can now be transfected at high
efficiencies. This considerably expands the range of cells in
which plasmid-based RNAi expression is feasible.
Thus, with nucleofection and its high transfection efficiency,
you can easily do both siRNA plasmids (as shown in the
example in Figs. 1 and 3) and siRNA duplexes (as illustrated by
the example in Fig. 2).
3. In rare cases, DNA toxicity may also be a concern. As cells are
considerably more tolerant of transfected RNA, for some cells
this may be the only way they can be efficiently transfected.
› page 14
› amaxa news # 5
Fig. 1: Knockdown effectiveness of different siRNAs to
SMYD3 (from Hamamoto et al., Nat. Cell Biol. 6(8), 731)
SMYD3 encodes a histone methyltransferase involved in the
proliferation of cancer cells. The hepatocellular carcinoma cell
line SNU475 was transfected by nucleofection with plasmids
expressing different SMYD3 siRNAs (1, 4, 12, and 14). Control
sequences were a 2bp-mismatch (mm) and scrambled
sequence (scr) of SMYD3-12, or siRNA targeted against eGFP.
(A) Western blot analysis of SMYD3 protein levels at 48h after
transfection; b-actin served as a loading control. Different
siRNAs lead to different levels of knockdown. (B) Effect
of SMYD3 siRNAs on cell growth measured with MTT proliferation assays 9 days after transfection and subsequent
incubation with G418 (the siRNA plasmids also contained
a neomycin-resistance cassette). The levels of knockdown
correspond with the effects on proliferation.
(Copyright 2004 by Nature Publishing Group. Reproduced
from Nature Cell Biology 6(8), 731 by copyright permission of
Nature Publishing Group and by permission of the authors.)
4
4
ments.
1. Include a characterized positive control.
ed
2
siRNA
Ri
t4
2
ra
m
Rit 42
2 4 day
Sc
siRNA:
of including appropriate controls in all experi-
bl
bl
A
Lu
cf
Sc .
ra
m
These observations underscore the importance
ed
› Hot topic
4 2 2 4 4 day
Rit 42
Rit 42
36B4
b-actin
mRNA
protein
2. Use a scrambled sequence of experimental
- Nocodazol
B
200
160
160
counts
120
Lucif.
-siRNA
5.92%
M1
80
40
0
3. Use the lowest siRNA concentration sufficient
0
0
0
160
counts
Scambled
siRNA
160
4.5%
M1
80
40
0
0
120
40
0
200 400 600 800 1000
0
200
160
160
4.71%
M1
80
40
0
0
200 400 600 800 1000
FL2-A
120
counts
counts
same target gene.
Rit42
siRNA
200 400 600 800 1000
FL2-A
200
120
5. Confirm results with second (or third) siRNA to
5.9%
M1
80
FL2-A
protein levels.
200 400 600 800 1000
FL2-A
200
120
4. Validate gene knockdown at both the RNA and
40
FL2-A
to elicit the desired reduction in gene expres-
5.06%
M1
80
200 400 600 800 1000
200
sion.
120
counts
sequence is not complementary to any other
gene in organism).
+ Nocodazol
200
counts
siRNA as negative control (ensure this
22.91%
M1
80
40
0
0
200 400 600 800 1000
FL2-A
6. If feasible, examine global changes in gene
expression.
7. If feasible, perform “rescue” experiments, e.g.
express an siRNA resistant form of the target
gene (e.g. containing silent mutation in middle
of siRNA binding site).
Time course of inhibition
It is often beneficial to measure the duration
Fig. 2: Knockdown of the p53 target gene Rit42 in normal human mammary
epithelial cells. (from Kim et al., J. Biol. Chem. 279(37), 38597)
(A) Cells were transfected by nucleofection with Rit42, scrambled-Rit42, or control
luciferase siRNA and cell lysates were extracted for Northern and Western blot analyses
at the indicated times following transfection. 36B4 and b-actin are included as loading
controls on the respective gels. (B) Effect of Rit42 suppression on cell population. Inhibition of Rit42 expression increased polyploid cell populations from ~5% to ~23% after
treatment of siRNA transfected cells with the spindle checkpoint inhibitor nocodazole,
as shown by FACS analysis after 48h. These findings suggest that Rit42 functions as a
mitotic checkpoint gene, ensuring cell division fidelity and maintenance of euploidy.
(Copyright 2004 by and reproduced with copyright permission of American Society For
Biochemistry & Molecular Biology)
of inhibition for both the target RNA and the
corresponding protein in order to compare with
A
observed downstream effects. Additionally, if
mRNA reduction is seen without a corresponding reduction in protein levels, it could mean
that protein turnover is particularly slow. Conversely, protein reduction in the absence of
B
mRNA reduction may indicate that the siRNA
is mediating its effects at the translation level as
is known for microRNA [3].
References
1. Van De Parre et al. Biochem Biophys Res Commun. (2005)
327:356-360.
2. Persengiev et al. RNA. (2004) 10:12-18.
3. Doench et al. Genes Dev. (2003) 17:438-442.
Fig. 3: Fat1 cadherin is required for tight cell-cell association and actin organization (from Tanoue and Takeichi, J. Cell Biol. 165(4), 517)
(A) Cells from the transformed mouse keratinocyte cell line PAM212 transfected with
Fat1 RNAi plasmid were doubly immunostained for Fat1 (red) and b-catenin (green) 2
days after transfection. More than 95% of the cells were transfected and showed a
reduction in the level of Fat1 protein. The pictures show areas with transfected and
untransfected cells side by side to illustrate the knockdown effects. Note the looser
cell-cell associations for Fat1-negative cells compared with those for the positive cells,
visualized by the b-catenin staining. (B) A similar culture immunostained for Fat1 (red)
and F-actin (green). Arrowheads point to junctional accumulation of actin fibers. Arrow
indicates the loss of F-actin-delineating cell junctions in Fat1-negative cells.
(Reproduced from The Journal of Cell Biology, 2004, 165(4), 517 by copyright
permission of The Rockefeller University Press and by permission of the authors.)
› page 15
› www.amaxa.com
› New product
new
Basic Nucleofector® Kits –
bringing new perspectives for
your research with primary
mammalian cells
amaxa’s new selection of Basic Nucleofector® Kits greatly enhances the range of primary mammalian cells you can efficiently transfect.
Now available:
The introduction of these kits allows you to optimize the transfection conditions of your primary
Basic Nucleofector Kits for:
mammalian cell type within just one experiment
›
›
›
›
›
using a remarkably simple procedure.
Endothelial cells
Epithelial cells
Fibroblasts
Just one Solution and five Programs away
Neural cells
from reaching transfection efficiencies of up
Smooth muscle cells
to 80%!
The strategy
1. Choose the Basic Nucleofector® Kit
for your primary mammalian cell
type e.g. Basic Nucleofector® Kit for
Epithelial Cells.
2. Test one Nucleofector Solution in combination with five different Nucleofector Programs.
E.g.:
Program
3. Identify the optimal Nucleofector
Program for best transfection efficiency and viability.
E.g.:
Transfection
efficiency (%) viability (%)
S-05
T-13
T-20
T-23
U-17
[k]
10
50
65
55
40
Ordering information
50
80
80
40
50
> > >
amaxa web information
Basic Nucleofector® Kit for Primary Mammalian Endothelial Cells
Cat. No.: VPI-1001
First results now available on
Basic Nucleofector® Kit for Primary Mammalian Fibroblasts
Cat. No.: VPI-1002
www.amaxa.com/celldatabase
Basic Nucleofector® Kit for Primary Mammalian Neural Cells
Cat. No.: VPI-1003
Basic Nucleofector® Kit for Primary Mammalian Smooth Muscle Cells Cat. No.: VPI-1004
Basic Nucleofector® Kit for Primary Mammalian Epithelial Cells
› page 16
Cat. No.: VPI-1005
› amaxa news # 5
› Application note
Mouse ES cells – a promising
tool in molecular genetics
Mouse embryonic stem (ES) cells were first isolated more then 20 years ago in 1981 (1, 2). Since then,
their unique properties and their potential in gene targeting has revolutionized molecular genetics.
Nucleofection as a powerful transfection technology for various cell types is also well suited for
gene delivery in mouse ES cells. Take a closer look and learn more about mouse ES cell nucleofection and the impact it can have on your research.
General introduction
Mouse ES cells are derived from the
inner cell mass of 3.5-day-old mouse
embryos. When ES cell lines are cultured
in the presence of leukemia inhibitory
factor (LIF) or on top of a layer of mitotically inactivated mouse embryonic
fibroblasts, they remain in an undifferentiated state and can proliferate indefinitely. ES cells are pluripotent. With
the appropriate stimuli provided in the
culture they can differentiate in almost
all tissue-specific cell lineages. These
include cell types from all three embryonic germ layers, such as cardiomyocytes, smooth muscle cells, hematopoietic
progenitors, hepatocytes, chondrocytes, melanocytes and so on (Figure 1).
With their unique characteristics mouse
ES cells are a promising tool for various
research applications and provide an
outstanding model to study diseases or
cell differentiation. In gene targeting
approaches, a DNA sequence of interest
is genetically engineered. It is then
transfected into ES cells and replaces
the wild-type sequence via homologous
recombination. The modified ES cell is
then injected into a blastocyst of a
foster mother and a mouse can be
generated with the modified gene in all
nucleated cells. When the mutation
results in inactivation of a specific
gene, a ‘knock-out’ mouse can be created, and the consequence of this genotype in a developing mouse can be
assessed. Gene targeting in mice is
often used for the generation of a
mouse model of human diseases, such
as Parkinson’s Disease, diabetes, chronic heart disease and multiple sclerosis.
A second promising approach involves
the differentiation of mouse ES cells in
vitro and the assessment of the regulation of cell differentiation in development. Transfection is used to either
modify cell differentiation or even trigger cell development along a certain
tissue lineage.
Nucleofection® of mouse ES cells
Meaningful research on differentiation
of ES cells in various cell lineages will
rely on the efficient gene transfer into
ES cell culture
Embryoid body
Ectoderm
Mesoderm
Endoderm
Skin cells
Cardiomyocytes
Pancreatic cell
Neurons
Skeletal muscle cells
Lung cells
Pigment cells
Kidney cells
Hepatoytes
Blood cells
Fig. 1: Pluripotent potential of murine ES cells.
› page 17
› www.amaxa.com
› Application note
References
1. Evans MJ. Mol
Biol Med
(1989)
6:557-565.
2. Martin GR.
Proc Natl Acad
Sci USA
(1981)
78:7634-7638.
Ordering information
Cat. No.: VPH-1001
q
into either diploid or tetraploid blastocysts (Boljahn et al., 2004).
To learn more on mouse ES cell
nucleofection simply fill in the reply
card in this newsletter and order a
copy of the application note or go to
www.amaxa.com/literature to download the pdf version.
Fig. 1: Comparison of nucleofection
and electroporation for transient
transfection of mouse ES cells Mouse
ES cells were transfected with a lacZ
reporter by nucleofection (A) or electroporation (BioRad Gene Pulser) (B) and
stained 48 hours after transfection for
transient lacZ expression (amaxa application note, Boljahn et al. 2004).
A
B
Supplementary information
amaxa application note
(Boljahn et al. 2004)
› Hot topic
Nucleofection® of stimulated
mouse T cells
Some of the research questions to be addres-
90
sed with the newly introduced Mouse T Cell
80
Nucleofector ® Kit involve the transfection of
70
stimulated cells. The data presented by the
60
DRFZ - Deutsches Rheuma-Forschungszentrum, Germany, show the successful nucleofection of mouse T cells that have previously been
stimulated. For more information on mouse T
cell transfection please go to pages 4-7 of this
newsletter.
% transfected CD4+ T cells
[k]
ES cells. As demonstrated by an amaxa
application note (Boljahn et al., 2004),
the Mouse ES Cell Nucleofector® Kit is
a well suited tool to assist you in
these experiments. Transient transfection of ES cells by nucleofection with
a lacZ reporter was found to be superior to standard electroporation. With
nucleofection a stronger and more
uniform protein expression was seen,
as demonstrated here for lacZ expression. In contrast, standard electroporation led to mostly mosaic lacZ
expression with a lower transfection
efficiency (Figure 1).
In addition, nucleofection has also
proven its usefulness in the generation
of chimeric mice. Viable and fertile
male and female mice were generated
by injection of nucleofected ES cells
50
40
30
20
10
0
GFP-PLL3.7
GFP-pEGFP-C1
hCD4-PCI
Example for transfection of stimulated mouse T cells. CD4+ mouse T cells isolated from spleens and lymph nodes of BALB/c
mice were stimulated for 22 h with 1.5 µg/ml aCD3 and 1.5 µg/ml aCD28 antibodies and transfected with either one of two different GFP expressing constructs (1 µg) or one hCD4 expressing vector (2 µg). 27 hours after nucleofection cells were analyzed for
GFP or hCD4 expression by flow cytometry. Transfection efficiency is given as % of transfected living CD4+ mouse T cells. Different efficiencies for the GFP expressing plasmids are due to different backbones used for each construct. (Data courtesy of
Andrej Mantei, Sascha Rutz, Alexander Scheffold, DRFZ- Deutsches Rheuma-Forschungszentrum, Berlin, Germany)
› page 18
› amaxa news # 5
› amaxa insights
amaxa Down Under
Dr. Tim Doran
Intergrated Sciences Team
“G’day” to our Australian distributor:
Integrated Sciences
Integrated Sciences’ ethos statement, “Our Strate-
These research initiatives have used Nucleofector
gy is Support,” captures the ongoing commitment
technology to introduce short hairpin RNA (shRNA)
to customers upon which it has built its business.
sequences. Dr. Doran’s group reports that Nucleo-
By selecting quality products and backing them with the highest
fection provides the ability to transfect cell lines
levels of scientific support and personal service, Integrated Science
including bovine macrophages and bovine kidney
has served the Australian market with distinction.
(MDBK) cells, which have proven difficult to trans-
That´s why we sought out Integrated Sciences, when amaxa wanted
fect in the past.
to bring Nucleofector technology to researchers in Australia.
The research group works with the PR8 strain of
Founded in 1983, Integrated Sciences is headquartered in Sydney
influenza A in MDCK (canine kidney) cells as part of
and maintains offices in Melbourne, Brisbane, and Adelaide. The
its antiviral research. These cells can be extremely
company takes great pride in the quality of its field representatives,
difficult to transfect efficiently with lipid-based rea-
molecular and cellular biologists, who embody its values. In combi-
gents; therefore, nucleofection has become the pre-
nation with a dedicated in-house staff of technical, service, business,
ferred transfection method. In a sample system using
and logistics specialists, the Integrated Sciences team delivers con-
green fluorescent protein (GFP) as a marker, Nucleo-
sistent high value to the scientific and diagnostics communities.
fector technology facilitated transfection of both a
GFP-encoding plasmid and a GFP-specific shRNA.
CSIRO Livestock Industries puts RNAi technology to work
Australia’s Commonwealth Scientific and Industrial Research Orga-
MDCK cells transfected using Nucleofector
nisation (CSIRO) is one of the world’s largest and most diverse
scientific global research organizations. CSIRO has been a consistent leader in the development of RNAi technology; in the early
pEGFP-N1
1990s Dr. Peter Waterhouse pioneered the expression of gene silencing double-stranded RNA (dsRNA) hairpins from DNA templates
pEGFP-N1 +
scrambled shRNA
pEGFP-N1 +
EGFP shRNA
MDCK cells transfected using lipid reagent
in plants (known as DNA-directed RNAi, or ddRNAi).
In the RNAi Technologies laboratory at CSIRO Livestock Industries,
project leader Dr. Tim Doran has been applying ddRNAi to functionpEGFP-N1
al genomic studies in cows and chickens, with a focus on immune
system function and production traits. His group is also working on
antiviral therapies using RNAi technology, using avian influenza in
chickens as a model system.
› page 19
pEGFP-N1 +
scrambled shRNA
pEGFP-N1 +
EGFP shRNA
Fig. 1 illustrates the highly efficient transfer and expression of GFP
sequences (compared with transfection using a lipid reagent),
followed by complete gene silencing using shRNA. In the words
of one researcher, “the pictures speak for themselves!” (Data
courtesy of Dr. Tim Doran, CSIRO Livestock Industries, Australia.)
› www.amaxa.com
› amaxa insights
R&D: Journey to the heart
of amaxa
Beginnings
An R&D-driven company
In the late 1990s, the study of gene function and
amaxa has now rapidly grown to a staff of 120 people, with re-
interactions between genes was advancing
search and development remaining at the heart of amaxa’s activi-
rapidly. For example, the availability of gene
ties. Now directed by Chief Scientific Officer Gregor Siebenkotten
“knock-out” and “knock-in” technology, using
and VP of Research and Development Titus Kretzschmar, the
pluripotent cells, rendered it possible to analyze
importance of R&D is reflected in amaxa’s aims: to develop
the effects of specific genes on developmental
and commercialize innovative gene transfer technologies and
pathways. However, studying these effects at the
products addressing unmet market needs, and to leverage them
cellular level remained difficult, in part because
for industrial, academic, and clinical applications.
of the inability to reliably introduce genes into
primary cells at high efficiency, while retaining
In addition to developing new products, and new protocols to
high cell viability.
support existing systems, the R&D group is a central part of the
amaxa team with multiple interfaces to various departments.
This roadblock was frustrating to Gregor Sieben-
Fruitful interactions with Marketing and Sales as well as Business
kotten and Rainer Christine, two researchers
Development do not only provide feedback on customer response
who were former colleagues at the University of
to new and existing products, and help pinpoint customer needs
Cologne, Germany. Realizing that this limitation
for new protocols, but also support the identification of emerging
was affecting research progress across multiple
techniques and technologies that help guide new product deve-
fields, they started amaxa in 1998, focusing their
lopment.
efforts on developing a reliable system for transfecting primary as well as other so-called “hard-
Furthermore, the R&D team interacts intensively with the Pro-
to-transfect” cell lines. The result of their work
duction Group to master the challenge of taking new products
was the introduction of the first Nucleofector
from the lab bench to manufacturing scale. Once a new method
system, with kits for specific cell types, in 2001.
or product is being developed, R&D works with Quality Assurance
› page 20
› amaxa news # 5
› amaxa insights
to establish appropriate specifications, and QA
for more types of primary human and animal
makes sure that only products of constantly high
cells. The recent introductions of innovative
quality are approved and released to our cus-
transfection kits for mouse T cells, of an expan-
tomers.
ded selection of protocols for cells used in cancer
research, and the further development of the uni-
Last but not least, R&D, Business Development,
que Nucleofector device resulting in the Nucleo-
and the Intellectual Property department have
fector II, exemplify the continuing success of the
established very close links to expand amaxa’s
technology.
patent portfolio.
The development of a Nucleofector system for
high-throughput applications offering unmatched
Current activities
flexibility is an example for an important new
amaxa’s research team is one of the world’s
project. Looking ahead, the R&D team is also
largest groups dedicated to the development of
working hard on the development of new pro-
non-viral methods for gene transfer into a wide
prietary technologies that promise to help you
range of cell types. The addition of talented,
address additional transfection challenges.
ambitious researchers has expanded the R&D
group’s energetic and creative mix of experiences,
Research and Development will continue to be
fostering an intense team spirit as well as conti-
at the heart of amaxa’s activities, as we address
nued excellence in product development and new
the transfection challenges in biological re-
research initiatives.
search.
The R&D team continues to expand and refine the
line of Nucleofector products, such as new kits
› page 21
› www.amaxa.com
› FAQ
FAQ - frequently asked questions
about Nucleofector® technology in immunology
›
›
What is the best method for detaching human
monocytes from the plate for assaying?
Can I also nucleofect mouse T cells
derived from lymph node or thymus?
›› We have had good results by incubating the cells in ice-cold PBS
›› Our Mouse T Cell Nucleofector® Kit has been
for 10 minutes and then rinsing the plates. Alternatively, detaching
optimized for mouse T cells from spleen. However,
the cells without medium change by gently pipetting up and down
other Nucleofector users have transfected mouse T
the cell suspension in the well will also work. Our experience has also
cells from lymph node or thymus successfully with
been that the cells are very sticky after only a few hours but will
somewhat lower transfection efficiency or viability.
detach more easily after 24 hours.
Especially for thymocytes, a higher mortality may
be seen as part of these cells may already be in an
›
How do you recommend to isolate lymphocytes from
apoptotic stage. When nucleofecting mouse T cells
mouse spleens? Is erythrocyte lysis required?
from organs other than spleen we recommend
›› We recommend cutting the spleen once and passing the tissue
using the same conditions as outlined in the Opti-
through a 100 µm cell strainer or a steel mesh using a plunger. The
mized Protocol for spleen cells.
cells are then flushed into a petri dish containing PBS. In order to
remove fat, cell debris and aggregates, the cells can further be pas-
›
sed through a 70 µm cell strainer or a cotton wool column. With this
Is the age of the mice important for my
mouse T cell nucleofection?
procedure, each spleen should yield approximately 5x10 -2x10 cells.
›› Yes. We recommend using mice between 6-15
We strongly recommend omitting the erythrocyte lysis step, since it
weeks. Using mouse T cells isolated from older
leads to a decrease in lymphocyte viability after nucleofection. Due
animals for nucleofection may result in much
to relatively low numbers of erythrocytes in the spleen cell prepara-
lower transfection efficiencies and/or viabilities.
7
8
tion (ratio of 1:2 leukocytes:erythrocytes compared to 1:1000 in
›
human peripheral blood), an erythrocyte lysis is not required.
Do the recovery media contained in the
Mouse T Cell and Human Monocyte
›
Nucleofector® Kits contain cytokines?
Do you recommend positive selection or depletion for
›› No, both post-nucleofection recovery media
the purification of human monocytes?
›› We only recommend the depletion method, e.g. using a MACS
do not contain cytokines or growth factors and
depletion kit. The advantage of depletion is that the monocytes
both media should not influence cell stimulation
are left untouched by antibodies during the process.
or differentiation. For mouse T cells, for example,
several experiments, some of which are outlined
›
Does it matter if the PBS used for monocyte enrichment
in this amaxa news (see pp. 4-8), have shown that
contains calcium and magnesium?
after nucleofection cells can be stimulated effi-
›› The PBS should be calcium and magnesium free to prevent clum2+
ciently in the recovery medium containing FCS.
2+
ping of cells. We routinely use PBS without Ca and without Mg .
amaxa web information
www.amaxa.com/faq
Scientific Support Europe/World
USA
phone
+49(0)221-99199-400
(240) 632-9110
fax
+49(0)221-99199-499
(240) 632-9112
e-mail
[email protected] [email protected]
More information needed?
It takes just one email, phone call or fax to obtain
› page 22
more information on the Nucleofector technology.
› amaxa news # 5
Just contact our Scientific Support Team.
› amaxa insights
Meet amaxa in 2005 at:
Please feel welcome to visit
Apr 2 – 6
amaxa´s booth at the following
Experimental Biology
San Diego, USA, booth no. 517, www.faseb.org/meetings/eb2005
meetings and shows this year!
Apr 11 – 12
Tumor Cell Biology Meeting
Lunteren, The Netherlands
Apr 17 – 20
AACR – 96th Annual Meeting of the American Association
for Cancer Research
Anaheim, USA, booth no. 276,
www.aacr.org/2005am/2005am.asp
May 17 – 20
7th Colloquium of the French Society for Neuroscience
Lille, France, www.neurosciences.asso.fr/Activites/colloques/SN05
Jun 1 – 5
ASGT – 8th Annual Meeting of the American Association
for Gene Therapy
St. Louis, USA, booth no. 415, www.asgt.org
Sep 3 – 6
ELSO 2005
Dresden, Germany, booth no. 31, www.elso.org
Sep 21 – 24
DGfI – German Society for Immunology
Kiel, Germany, www.immunologie.de
Oct 1 – 5
DGHO – German Society for Haematology and Oncology
Hannover, Germany, www.dgho.de
Nov 12 – 16
Neuroscience 2005 – 35th Annual Meeting of the Society
for Neuroscience
Washington, USA, www.sfn.org
Dec 10 – 14
ASCB – 45th Annual Meeting of the American Society
for Cell Biology
San Francisco, USA, www.ascb.org
www.amaxa.com/meetings
amaxa´s Nucleofector® process, Nucleofector® device and Nucleofector® solutions are covered by PCT
applications PCT/EP01/07348, PCT/DE 02/01489, PCT/DE 02/01483, other pending patents and domestic
or foreign applications corresponding thereto.
amaxa, Nucleofector, nucleofection, maxGFP and maxFP are trademarks of amaxa GmbH.
› page 23
› www.amaxa.com
amaxa GmbH
Nattermannallee 1
50829 Koeln
Germany
amaxa Inc.
205 Perry Parkway
Suite 7
Gaithersburg, MD 20877
USA
Scientific Support
Europe/World
phone +49(0)221-99199-400
fax
+49(0)221-99199-499
e-mail [email protected]
Scientific Support
USA
phone (240) 632-9110
fax
(240) 632-9112
e-mail [email protected]
Register now to receive
amaxa´s new catalog 2005!
With detailed product descriptions as well as useful additional information on all of amaxa's
products - it's definitely worth having an amaxa
catalog in reach!
WAA-1001_05
www.amaxa.com/catalog2005
› w w w. a m a x a . c o m