amaxa news #10 - Lonza AG

# 10
www.amaxa.com
amaxa news
Functionality Matters
Nucleofection ® Provides Superior Preservation of Functionality
New Products
amaxa’s Portfolio for Mouse Dendritic Cells
Basic Neuron SCN Nucleofector ® Kit
The Latest Nucleofector ® Kits for Stem Cells
amaxa Meets Promega
Nucleofector ® Technology and Luciferase Reporter Assays
Save Time – Achieve Your Objectives
Introducing: amaxa’s Transfection Services
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Cell Line …
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nucleofection ® conditions of your
cell line?
Take advantage of the reduced
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Nucleofector ® Kit now and realize
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Editorial
“In this study, the amaxa nucleofection ® methodology was used
to achieve transfection efficiencies of up to 65%. The transfection
procedure per se had neither appreciable effects on the activation
state of cells nor on their sensitivity to exogenous TCR stimulation.
This experimental set-up therefore allowed a first functional
analysis of HIV-1 Nef in resting CD4 + T cells.”
Keppler et al. (2006) Journal of Leukocyte Biology, 79:616-627.
Dear Scientist,
For researchers working in the field of gene transfer, efficiency and
viability are of utmost importance. To us, however, it has always
been clear that there existed another core interest – the maintenance
of biochemical cell functionality post transfection. That is
why we chose functionality as a key topic for amaxa news # 10.
Read the Application Note on pages 10 and 11 to learn how
nucleofection ® is optimized to preserve superior cell functionality.
Numerous publications have also dealt with this observation.
The above quote clearly underlines the outstanding status of
Nucleofector ® Technology compared to other transfection methods.
In order to develop applications that will provide cell biologists
broader access to monitor cell signaling events in difficult-totransfect
cells, amaxa has teamed up with Promega. More
information on the first results of this cooperation can be found on
pages 6 and 7.
Of course news # 10 also keeps you updated with the latest additions
to our product portfolio. The last months have seen the
introduction of new Kits and Protocols for Mouse Dendritic Cells
(page 4), Neurons (page 5) and Stem Cells (page 14).
Best regards, Rainer Christine
CEO

Editor
Wolfgang Kintzel
Product News
Product News
Editorial board
Oliver Gresch, Katrin Höck, Ken Dwyer,
Gerhard Muster, Claus-Dietmar Pein,
Volker Vogel, Martina Mohrs,
Kevin Mobbs, Andrea Toell
Production coordinator
Benno Limberg
Hot Topic
Application Note
Application Note
Product News
Product News
Product News
Product News
Application Note
amaxa Insights
Hot Topic
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amaxa AG
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Tel: +49(0)221-99199-0
Fax: +49(0)221-99199-111
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› Table of Contents
› Page 3 › www.amaxa.com
4 amaxa’s Product Portfolio for the Nucleofection ® of
Mouse Dendritic Cells
5 Basic Neuron SCN Nucleofector ® Kit:
Transfect Only What You Need for One Coverslip!
6 Nucleofection ® Enables Luciferase Reporter Assays in
Primary Cells and Virtually Any Cell Line
8 Down-regulation of a Key Component of the RNA Localization
Machinery in Hippocampal Neurons
Manuel Zeitelhofer and Ralf Dahm, Division of Neuronal Cell Biology,
Center for Brain Research, Medical University of Vienna, Vienna, Austria
10 Nucleofection ® – Combining High Transfection Performance
with Superior Preservation of Functionality
12 Higher Efficiencies for Jurkat Cells Than Ever Before
13 96-well Shuttle ® Protocols and Kits – Human T Cells, HepG2, SH-SY5Y
14 From Mouse to Human – The Latest Nucleofector ® Kits for Stem Cells
15 pmaxCloning Vector – Strong Expression of Your Individual cDNA
16 Achieving Efficient Delivery of Morpholino Oligos
with Nucleofection ®
Shannon T. Knuth, Jon D. Moulton and Paul A. Morcos, Biology and Customer
Support, Gene Tools, LLC, Philomath, Oregon, USA
18 amaxa’s QA and Production Team
20 Achieve Your Research Objectives with amaxa Transfection Services
21 FAQs on Nucleofection ® and Preservation of Functionality
22 amaxa’s Distributor Overview
23 Meet amaxa in 2008
24 amaxa’s Webinars 2008
Please note that to date amaxa’s products are intended to be used for research and investigational use by
professionals only and shall under no circumstances be used for diagnostic purposes, for testing or treatment
in humans.
The Nucleofector ® Technology, comprising Nucleofection ® Process, Nucleofector ® Device, Nucleofector ®
Solutions, Nucleofector ® 96-well Shuttle ® System and Nucleocuvette ® plates and modules is covered by patent
and/or patent-pending rights owned by amaxa AG.
amaxa, gene transfer begins here, HiFect, maxFP, maxGFP, Nucleocuvette, nucleofection, Nucleofector,
pmaxCloning and 96-well Shuttle are either registered trademarks or trademarks of amaxa AG in Germany
and/or the U.S. and/or other countries.
ATCC ® and the ATCC Catalog Marks are trademarks of ATCC used under License; BD FACSCalibur is a
trademark of Becton, Dickinson and Company; Dharmacon, DharmaFECT, siARRAY, siGENOME, SMARTpool,
and siCONTROL are registered trademarks of Dharmacon Inc.; ON-TARGETplus is a trademark of Dharmacon Inc.;
Qiagen and EndoFree ® are trademarks of Qiagen; QuantiGene ® is a trademark of Panomics; CellTiter-Blue ® ,
CellTiter-Glo ® and Apo-ONE ® are trademarks of Promega; Pre-MiR is a trademark of Ambion; Tecan and
Freedom EVO ® are trademarks of Tecan Group Ltd.
Other product and company names mentioned herein are the trademarks of their respective owners.
Design
vierviertel – Agentur für Kommunikationsdesign GmbH
info@vierviertel.com / www.vierviertel.com
© amaxa AG, January/2008. All rights reserved. Printed in Germany.

[k]
› Product News
new
Now Completed – amaxa’s Product
Portfolio for the Nucleofection ® of
Mouse Dendritic Cells
Mouse dendritic cells (DC) are a key element in the mammalian immune system, thus understanding
their role is of vital interest for academic research groups and industry. With one Nucleofector ® Kit
and two 96-well Shuttle ® Kits optimized for transfection of mouse dendritic cells, amaxa offers a
unique, ready-to-use product portfolio.
Key benefits › Up to 59% transfection efficiency
100
90
80
70
60
50
40
30
20
10
0
%
96-well Shuttle ®
› More than 90% cell viability
› Excellent preservation of functionality (see also page 10 + 11)
› Low cell numbers required
(2.5 - 5 x 104 cells for the 96-well Shuttle ® , 2.5 x 105 for the standard Nucleofector ® )
Efficiency
Nucleofector ®
Transfection performance.
Mouse DC (Balb/C) were transfected according to the
appropriate Optimized Protocol for nucleofection ® using
pmaxGFP. Cells were analyzed 24 hours post nucleofection ®
by flow cytometry for maxGFP ® expression and viability. Cell
viability is given in percent compared to non-transfected
control.
The Nucleofector ® Kit for mouse DCs is only compatible with Nucleofector ® II Devices (carrying software version S3-7
and higher for the Nucleofector ® II and version S4-4 and higher for Nucleofector ® II, serial version ”S“).
› Page 4 › amaxa news # 10
Functionality post nucleofection ® .
The graph displays functionality of immature mouse DC
(Balb/C mice) post nucleofection ® (Sample) on standard
Nucleofector ® and 96-well Shuttle ® . Analyzed was their ability
to secrete IL-6. Therefore, 2 hours post nucleofection ® , cells
were stimulated by LPS. 22 hours later, functionality was
analyzed by IL-6 specific ELISA and is given in percent compared
to non-transfected control (Control).
Ordering information Mouse Dendritic Cell Nucleofector ® Kit 25 reactions Cat. No.: VPA-1011
Mouse Dendritic Cell (immature) 96-well Nucleofector ® Kit 96 reactions Cat. No.: VHPA-1011
Mouse Dendritic Cell (mature) 96-well Nucleofector ® Kit 96 reactions Cat. No.: VHPA-1012
Mouse Dendritic Cell (immature) 96-well Nucleofector ® Kit 960 reactions Cat. No.: VHPA-2011
Mouse Dendritic Cell (mature) 96-well Nucleofector ® Kit 960 reactions Cat. No.: VHPA-2012
amaxa web information For further information, please contact your local representative or visit www.amaxa.com/mouseDC
Viability
100
90
80
70
60
50
40
30
20
10
0
Mouse DC functionality
%
96-well Shuttle ®
Control Sample
Nucleofector ®

new
› Product News
Basic Neuron SCN Nucleofector ® Kit:
Transfect Only What You Need for One
Coverslip!
[k]
amaxa’s new Basic Neuron Small Cell Number (SCN) Nucleofector ® Kit makes high efficiency transfection
possible even for extremely low cell numbers. Now you can transfect just the number of cells needed
for later culture and analysis (e.g., microscopic analyses of neuronal growth and morphology). Benefit
from a drastic reduction in both number of donor animals and amount of preparation time. The new,
optimized SCN Nucleofector ® Solution, SCN cuvettes, programs and optimized protocols make
transfection efficiencies of up to 60% possible with as little as 15,000 cells per nucleofection ® .
› Transfect as little as 15,000 cells per nucleofection ® –
More experiments with fewer donor animals
› Excellent non-viral transfection efficiencies and viabilities –
50% and higher even for very difficult-to-transfect neurons like DRGs
› Suitable for a broad range of neurons –
Easy optimization for your neuron of interest
Average transfection efficiencies and viabilities of
embryonal rat hippocampal neurons (n = 3).
20,000 and 50,000 freshly isolated rat hippocampal neurons
(E17) were transfected, cultured and analyzed as described
in amaxa’s Optimized Protocol for nucleofection ® . Viability
was determined as number of living cells compared to
non-transfected control.
(Data courtesy of M. Kiebler, Department of Neuronal Cell Biology,
Medical University of Vienna, Vienna, Austria).
SCN nucleofection ® of freshly isolated mouse Dorsal Root
Ganglion (DRG) cells.
15,000 DRGs (E15.5) were transfected with Nucleofector ® SCN
Basic Neuro Program 6 and 0.4 µg pmaxGFP ® (green, B) and
seeded on a coated coverslip. 24 hours post nucleofection ® ,
cells were fixed and immunostained for b -tubulin III
(red, neuronal marker, A). In this experiment, transfection
efficiency as determined by fluorescence microscopy is
approximately 60%.
(Data courtesy of B. Eickholt, MRC Centre for Developmental
Neurobiology, King‘s College, London, United Kingdom).
Ordering information Basic Neuron SCN Nucleofector ® Kit* Cat. No.: VSPI-1003
Nucleofector ® II Device, Serial Version “S” Cat. No.: AAD-1001S
*Due to the innovative pulse programs which are applied, SCN Kits are compatible with Nucleofector ® II Devices, serial version “S” only. Please make sure
that you have this version of device available by checking the information at www.amaxa.com/program-update or contacting our Scientific Support. For
customers with an older version of the Nucleofector ® Device who want to use SCN Kits, amaxa offers a trade-in program with very attractive conditions.
80
60
40
20
0
%
Transfection efficiency
A B
amaxa web information www.amaxa.com/basic-neuron-scn.html
› Page 5 › www.amaxa.com
Viability
5 x 104 2 x 10
Cells
4 Cells

› Hot Topic
Introduction
Nucleofection ® Enables Luciferase
Reporter Assays in Primary Cells
and Virtually Any Cell Line
Promega – First-class Luciferase Reporter Gene Vectors and Assays
› Reporter gene expression vectors for Firefly and Renilla luciferases
› All common selectable markers available
› Choice of different vectors containing no promoters, minimal promoters or promoters and response elements
› Control of reporter protein stability using Rapid Response technology
› Assay reagents for the detection of single or dual Firefly and Renilla luciferase activity
› Highly sensitive, robust and homogeneous assays
› Available as ‘flash’ or ‘glow’ type assay format
› Ideally suited for high-throughput applications
Reporter gene technologies are powerful tools
to study gene expression, regulation and complex
cellular processes such as gene networks.
Successful reporter gene expression requires
efficient delivery of state-of-the-art expression
vectors into appropriate cells. In addition, fast,
reliable and sensitive reporter assays are needed
to obtain meaningful results. amaxa’s non-viral
Nucleofector ® Technology is ideally suited for
transfection of primary cells and difficult-totransfect
cell lines. It provides easy and reproducibly
efficient transfection of virtually any cell
type in single reactions or up to 96-wells at a
time. In combination with Promega’s luciferase
reporter technology, it leads to rapid and
high expression within a few hours. Promega’s
luciferase reporter vectors and assays represent
the gold standard for highly sensitive, simple
and flexible reporter gene studies. In the past
few years, new features were developed enabling
the choice of selectable markers, different
› Page 6 › amaxa news # 10
promoters and essential control of protein stability. In addition,
robust and homogeneous assays are available that allow for
usage in high-throughput applications. Thus, these two technologies
complement one another perfectly for such diverse uses
as measurement of transcriptional activity, RNAi-mediated gene
regulation and secondary screenings using difficult-to-transfect
cell types such as primary cells.
Nucleofection ® of Luciferase Reporter Vectors
into Primary Cells and Suspension Cell Lines
Influence of vector constructs on luciferase expression
Successful gene expression experiments are influenced by
numerous factors such as cell type, transfection method, the
expression vector, and analysis method. Constructs containing
the CMV promoter usually lead to stronger gene expression compared
to vectors containing the SV40 promoter in mammalian
cells, but there are exceptions such as BHK-21 cells. 1 In addition,
DNA sequences like the polyadenylation signal, enhancer, Kozak
sequence or introns may have an influence on gene expression
even if two expression plasmids contain the same promoter.

› Hot Topic
Kinetics of luciferase expression after nucleofection ® differ from other reporter genes
Unlike conventional transfection methods, such as lipofection, nucleofection ® delivers substrates
not only into the cytoplasm but also into the nucleus of the cell, resulting in a reduced time to expression.
It is highly recommended to perform luciferase analysis 6 - 16 hours post nucleofection ® , whereas
the optimal analysis time point for b –gal or GFP expression is 10 - 48 hours post nucleofection ® .
Nucleofection ® and Luciferase Assays Offer a Wide Range of Applications
Promoter studies by co-transfection of 4 expression vectors using nucleofection ®
Overexpression of HMGA1 proteins is a constant feature in human carcinomas and plays a critical
role in controlling growth of NK/T cell lymphocytes. By co-transfection of 4 expression constructs,
Fedele et al. have shown that HMGA1 proteins and transcription factor NF- B exert a synergistic
k
effect on the activity of the IL-15 promoter in NK-T leukemic cell line DERL-7. 2 They investigated the
functional consequences of HMGA1-binding on IL-15 promotor activity by transfecting a reporter
vector in which luciferase expression is driven by the IL-15 promoter (pGL3 IL-15) with cDNAs expressing
HMGA1 and NF- B p65 into the DERL-7 NK-T leukemic cell line (Figure 1).
k
Figure 1: HMGA1 and NF- k B exert a
synergistic effect on the promoter
activity of IL-15.
Murine NK-T leukemic cell line DERL-7
was transfected with the IL-15 promoter
reporter vector alone, or co-transfected
with 10 µg of NF- k B p65, or with 10 µg of
both NF- k B p65 and HMGA1 expression
vectors using Nucleofector ® Cell Line
Kit V and Nucleofector ® Program O-017.
Furthermore, a b -gal expression plasmid
was used for normalization of the transfection
efficiency. Luciferase activity
was measured 4 hours post nucleofection ® .
(Data reproduced from Fedele et al.
(2005) Oncogene 24:3427-3435).
Conclusions and Outlook
The combination of Promega’s luciferase vectors and assays and the amaxa Nucleofector ® Technology
provide unique tools to examine gene expression, RNAi-mediated gene regulation and many further
applications in cell biology. These technologies allow studies in the scientifically most relevant
primary cells and lead to more meaningful results with reliable and sensitive assays.
References
1. Liu et al. (1997) Anal Biochem 246(1): 150-152.
2. Fedele et al. (2005) Oncogene 24:3427-3435.
Promega and Rapid Response are trademarks of Promega Corporation.
› Page 7 › www.amaxa.com
12
10
8
6
4
2
0
Luciferase activity (arbitrary units)
_
+ +
_ _
pGL3
+
pCMV- b -gal
_
+ +
_ _
pGL3 IL-15
+
NF B p65
k
HMGA1

› Application Note
Down-regulation of a Key Component
of the RNA Localization Machinery
in Hippocampal Neurons
Manuel Zeitelhofer and Ralf Dahm, Division of Neuronal Cell Biology, Center for Brain Research,
Medical University of Vienna, Vienna, Austria
The localization of mRNAs is an efficient way to target proteins to specific regions in a cell. In mammalian neurons, RNA localization is involved in
compartmentalizing the cell during differentiation and is assumed to play an important role in synaptic plasticity. RNAs are packaged together with
RNA-binding proteins into transport-competent ribonucleoprotein particles (RNPs) and transported into dendrites. The RNA-binding protein Staufen2
(Stau2) is a key component of RNPs and implicated in dendritic RNA transport. In this study, Stau2 was successfully down-regulated by RNA interference
(RNAi). The knockdown efficiency of Stau2 expression was assessed after nucleofection ® of shRNA constructs targeting Stau2 mRNA. In contrast to
other transfection methods, the high transfection efficiencies attainable with the nucleofection ® technique allowed the determination of the extent of
Stau2 protein down-regulation by quantitative western blot analysis.
Introduction
The localization of mRNAs is an efficient
way to target gene products to specific
regions in a cell. It occurs in a wide range
of organisms and cell types 1, 2 . During
Drosophila development, for example,
localized mRNAs act as cell fate determinants
and thereby specify the body
axes 3 . In addition to its role in development,
RNA localization also occurs in
differentiated cells. In migrating cells, for
instance, mRNAs are localized to the
leading edges, thereby enabling directed
movement. This plays a role in wound
closure when fibroblasts migrate into the
lesion or when cancer cells metastasize.
Polarized cells, such as neurons, display
a functional compartmentalization of the
cytoplasm. The cell body is molecularly
distinct from the axon and the dendrites.
RNA localization is involved in compartmentalizing
neurons during differentiation
and it is assumed to also play
an important role in synaptic plasticity,
the experience-dependent remodeling
of synapses that forms the basis of
learning and memory 4 .
How is RNA localization achieved?
Localized RNAs contain cis-acting
sequence elements, often within the
3’-untranslated region (3’-UTR), which
are recognized by proteins (trans-acting
factors). The mRNAs and trans-acting
factors are packaged into ribonucleoprotein
particles (RNPs). These RNPs
are then transported to and retained
by sites of local synthesis 5 . During transport,
the RNAs must be kept translationally
silenced to prevent the ectopic
expression of the corresponding protein.
At their destinations, this translational
block can be lifted by signals inducing
translation 6 . A central hypothesis assu-
mes that translation is only activated at
synapses where synaptic plasticity has
been induced.
In this study, the nucleofection ® technique
was used to assess the efficiency of
down-regulation of Staufen2 (Stau2) in
hippocampal neurons. Stau2 is one
of the key components implicated in
dendritic RNA localization. In Drosophila,
Staufen is required for mRNA localization
in the oocyte 7 and in neuroblasts 7 .
In mammals, it is hypothesized that
Stau2 is important in the transport of
RNAs into dendrites of mature hippocampal
neurons. To assess the consequences
of a loss of Stau2 on dendritic
RNA localization as well as the morphology
of dendrites and dendritic spines,
Stau2 was down-regulated by RNAi
in primary cultures of hippocampal
neurons 8 . In order to obtain quantitative
information on the extent of down-regulation
by western blot analysis, it was
crucial that a large proportion of hippocampal
neurons was transfected by
nucleofection ® .
Material and Methods
Primary hippocampal neuron culture
Neurons were isolated from the hippocampi
of 17 day-old embryonic (E17)
rat brains and cultured as described 9 .
For nucleofection ® experiments, neurons
were immediately used after dissociation.
For the assessment of dendritic
spine phenotypes and for fluorescence
in situ hybridization (FISH) experiments,
cells were used after 15 days in vitro (DIV).
Nucleofection ® and western blot
analysis
After dissociation, neurons were cotransfected
with an expression vector
› Page 8 › amaxa news # 10
encoding citrine and shRNA constructs
(pSUPER, OligoEngine 10 ) against
Stau2, mismatch Staufen2 (misStau2),
and Septin7 (Sept7; CDC10). Cells were
plated onto 6 cm cell culture dishes
and lysed after 3 days of expression.
The knockdown efficiency was assessed
by western blot analysis.
Immunostaining
The following antibodies were used:
an affinity-purified rabbit anti-Stau1
antibody (1 µg/ml) and an affinity-purified
rabbit anti-Stau2 antibody (1 µg/ml).
Fluorophore-coupled phalloidin was used
to label F-actin. Immunostaining experiments
were performed as described 11 .
Results
Stau2 was down-regulated in hippocampal
neurons by nucleofection ® of shRNA
constructs to assess its function during
neuronal differentiation and in mature
neurons. To evaluate the success of the
down-regulation, western blot analysis
of the transfected neurons was performed.
As neurons are post-mitotic cells,
they are difficult to transfect. Conventional
transfection methods like the
calcium phosphate (CaPi) method have
very poor transfection efficiencies for
subsequent biochemical analyses and
virus-based methods are too time-consuming
and costly 12 . The nucleofection ®
technique overcomes these limitations.
Primary hippocampal neurons were
co-transfected with an expression vector
encoding citrine and shRNA constructs
(pSUPER) against Stau2, mis-Stau2,
and Septin7. First, the transfection
efficiency was evaluated. To this aim,
transfected neurons were fixed 3 days
post nucleofection ® and the percentage

› Application Note
A
of transfected cells quantified (Figure
1A, B). We reached transfection efficiencies
of approximately 60% that allowed
to perform biochemical experiments.
Importantly the transfected neurons
developed normally (Figure 1C, D). This
allowed us to quantitatively downregulate
Stau2 in neurons via RNAi. The
levels and the specificity of Stau2 downregulation
were assessed via western
blot analysis (Figure 2A). The protein
levels of Stau2 were significantly downregulated
in shRNA transfected cells
whereas the levels of the Stau2 paralogue
Stau1 as well as that of the unrelated
protein Septin7 remained unchanged.
Moreover, nucleofection ® with mis-Stau2
did not affect Stau2 protein levels,
indicating that the down-regulation of
Stau2 is specific.
The extent of down-regulation in mature
neurons was further controlled by
immunocytochemistry (Figure 2B). 15 DIV
neurons were co-transfected using the
CaPi method with an expression vector
encoding ECFP together with pSUPER
vectors against Stau2, misStau2 and
B
C D
o
o
A B
o
¬
Figure 1: Hippocampal neurons are transfected efficiently via nucleofection ® .
Hippocampal neurons were transfected with an shRNA construct against Stau2 and
cultured in 6 cm cell culture dishes. After 3 DIV, neurons were fixed and analyzed to
examine transfection efficiencies (B). The transfection efficiency reached up to
60%. Neurons displayed normal morphology upon nucleofection ® (A). Higher
magnification phase contrast (C) and fluorescence images (D) show the integrity of
the transfected neurons. The arrow shows an outgrowing axon and the arrowheads
indicate extending neurites with the typical growth cones at their tips. The inset in
C shows the corresponding DAPI stained nucleus of the transfected neuron.
RFP, respectively, and immunostained
with Stau2 and Stau1 antibodies. Stau2
staining was substantially reduced in
neurons transfected with the shStau2
plasmid but was abundant in neurons
transfected with mis-Stau2 or RFP plasmid.
The down-regulation of Stau2 did
not affect Stau1 staining.
Interestingly, down-regulation of Stau2
caused a rearrangement of the actin
cytoskeleton, which plays an important
role in the maintenance and plasticity of
dendritic spines. This effect was mirrored
by changes in the morphology of dendritic
spines from their characteristic mushroom-like
shape to filopodia. In addition,
it could be observed that the levels of
PSD95, a key component of the postsynaptic
density, were reduced in Stau2
down-regulated neurons, indicating that
there are fewer functional synapses
than in normal cells. This hypothesis
was borne out by electrophysiological
recordings that indicate a reduction in
synaptic transmission in Stau2 knockdown
neurons 8 .
As Stau2 has been implicated in dendritic
› Page 9 › www.amaxa.com
mRNA transport and since the actin
cytoskeleton is rearranged upon Stau2
down-regulation, it was further tested
whether the levels of -actin mRNA
b
are changed in Stau2 down-regulated
neurons. It was indeed observed that the
-actin mRNA levels were reduced by
b
approximately 37%, indicating that
Stau2 is important for the transport of
this mRNA into dendrites 8 .
Discussion
The Nucleofector ® Technology is ideally
suited to perform RNAi studies in postmitotic
cells, such as hippocampal neurons.
In contrast to other transfection
methods, the high transfection efficiencies
attainable with the nucleofection ®
technique allow a proper analysis of the
levels of Stau2 protein down-regulation
by quantitative western blot analysis.
In summary, this study showed that
the down-regulation of Stau2 causes
changes in dendrite morphology and
suggests a role of Stau2 in the transport
of b -actin mRNA into dendrites of hippocampal
neurons.
mock unr. mis Stau2 +siStau2
+si-RFP +mis +siStau2
Stau2
Calnexin
Stau1
Calnexin
*
ECFP ECFP ECFP ECFP
*
Stau2 Stau2 Stau2 Stau1
Figure 2: Assessment of the extent of down-regulation of Stau2.
(A) Western blot analysis of hippocampal neurons transfected using the Nucleofector ® Technology. Neurons were co-transfected
with a plasmid expressing citrine fluorescent protein (lane 1, mock) and shRNA-expressing plasmids against Septin7 (lane 2, labelled
unr.), mismatch Stau2 (lane 3, mis) and Stau2 (lane 4, siStau2). Cells were lysed after 3 days of expression and processed for
western blot analysis. The levels of the three Stau2 isoforms (lines indicate 62, 59 and 52 kD) were significantly down-regulated only
in cells transfected with the shRNA plasmid siStau2 (lane 4). Calnexin served as internal loading control. The levels of Stau1 did not
change upon down-regulation of Stau2. (B) Down-regulation of Stau2 in mature neurons. 15 DIV neurons were co-transfected with
the following constructs: siStaufen2-2 (siStau2), si-RFP or mismatch Staufen2 (mis) pSUPER vectors together with ECFP (green).
Neurons were stained 3 days after transfection with anti-Stau2 or anti-Stau1 antibodies (red). The Stau2 signal was strongly reduced
in both the cell body (asterisk) and dendrites of transfected neurons (green) compared with untransfected neurons. By contrast,
expression of misStau2 or si-RFP did not alter the Stau2 levels. Down-regulation of Stau2 also did not affect Stau1 expression. Scale
bar: 10 µm. (Figure 2a reproduced from (2006) Journal of Cell Biology 172:221-231. Copyright 2006 Rockefeller University Press.)
**
**
¬
¬
References
1. Kloc, Zearfoss
et al. (2002)
Cell
108(4): 533-44.
2. Jansen (2001)
Nat Rev Mol Cell Biol
2(4): 247-56.
3. St Johnston (2005)
Nat Rev Mol Cell Biol
6(5): 363-75.
4. Sutton and
Schuman (2006)
Cell
127(1): 49-58.
5. Martin and Zukin
(2006)
J Neurosci
26(27): 7131-4.
6. Hüttelmaier
et al. (2005)
Nature
438(7067):512-5.
7. Broadus,
Fuerstenberg
et al. (1998)
Nature 391(6669):
792-5.
8. Goetze, Tuebing
et al. (2006)
J Cell Biol
172(2): 221-31.
9. Zeitelhofer, Vessey
et al. (2007)
Nat Protoc
2(7): 1692-704.
10. Brummelkamp,
Bernards
et al. (2002)
Science
296(5567): 550-3.
11. Goetze, Grunewald
et al. (2004)
J Neurobiol
60(4): 517-25.
12. Dahm, Zeitelhofer,
Götze, Kiebler and
Macchi
(2008)
Methods
in Cell Biology
(in press)

› Application Note
Nucleofection ® –
Combining High Transfection
Performance with Superior
Preservation of Functionality
Nucleofection ® has become the method of choice whenever transfection of primary cells or hard-totransfect
cell lines is required. Here we show that nucleofection ® of frequently used primary cells
(mouse dendritic cells, human macrophages and human T cells) results in highly efficient transfer of
DNA and other substrates while at the same time maintaining excellent cell viability and post
transfection functionality. This combination of benefits makes nucleofection ® superior to other
transfection methods.
Introduction
For some primary cells, transient transfection
can be achieved by using
classical transfection methods, like
lipofection. However, none of the classical
transfection methods gain high
transfection rates combined with low
post transfection mortality and a good
preservation of cell-specific functionality.
With the Nucleofector ® Technology,
amaxa offers the first non-viral easyto-use
transfection method unifying
these three aspects. Great care is taken
during amaxa’s optimization of the
nucleofection ® parameters to further
minimize the effects of the transfection
process on cell functionality. This
includes the verification of cellular
functionality utilizing frequently used
cell-specific assays early in the developmental
process.
This short report focuses on three frequently
used, hard-to-transfect primary
Table 1
cells (primary mouse dendritic cells,
primary human macrophages and
primary human T cells) proving the high
degree of post nucleofection ® functionality
that can only be reached using
nucleofection ® .
Results and Discussion
The experiments presented in this
article have either been performed
on the standard Nucleofector ® (mouse
dendritic cells) or the high throughput
96-well Shuttle ® Device (human
macrophages and human T cells) using
the individual primary cell-specific
Nucleofector ® Kit or 96-well Nucleofector ®
Kits. Each primary cell-specific kit has
been optimized to perfection by amaxa’s
team of R&D scientists. This resulted
in excellent transfection efficiencies
while maintaining cell functionality.
Cells were transfected with pmaxGFP
› Page 10 › amaxa news # 10
using the parameters carefully optimized
for the primary cell and analyzed
24 to 48 hours post nucleofection ® by
flow cytometry for maxGFP ® expression.
Viability was either measured by
propidium iodide (PI) staining or using
microtiter plate based cell viability
assays (CellTiter-Glo ® , Promega). Viability
values are given in percent
compared to the non-transfected
control. Functionality of the cells post
nucleofection ® was tested by frequently
used cell-specific assays as described
below.
Mouse dendritic cells
Nucleofection ® of mouse dendritic cells
resulted in high transfection efficiency
(59%) and high cell viability (93%)
(Table 1, immature mouse dendritic
cells, strain BALB/c). In parallel, the
optimized parameters enabled an
Cell Nucleofector ® Kit used Program Transfection Cell
efficiency viability
Mouse Dendritic Cells Mouse Dendritic Cell Nucleofector ® Kit Y-001 59% (±3%) 93% (±7%)
Human Macrophages Human Macrophage 96-well Nucleofector ® Kit DP-148 42% (±2%) 60% (±6%)
Human T Cells, resting Human T Cell 96-well Nucleofector ® Kit FI-115 80% (±6%) 53% (±7%)
Human T Cells, resting Human T Cell 96-well Nucleofector ® Kit EO-115* 68% (±5%) 79% (±8%)
Human T Cells, stimulated Human T Cell 96-well Nucleofector ® Kit EO-115 70 % (±3%) 60% (±6%)
Transfection efficiency and viability 24 hours post nucleofection ® .
Cells were transfected according to the appropriate Optimized Protocols for nucleofection ® using pmaxGFP. Cells were analyzed
24 hours post nucleofection ® by flow cytometry. Cell viability is given in percent compared to non-transfected control (cells
handled as described in the Optimized Protocol but not treated with DNA or nucleofection ® program). For human T cells, two
programs were optimized giving either superior transfection efficiency (FI-115) or optimal post nucleofection ® functionality (EO-115).
Data are representative of 48 - 96 experiments. *Functionality data for resting human T cells are only shown for the program EO-115.

› Application Note
Percent functionality
100
90
80
70
60
50
40
30
20
10
0
Functionality post nucleofection ®
Nucleofector ®
Mouse Dendritic Cells /
IL-6 secretion
Figure 1: Cell functionality post nucleofection ® .
The bar graph displays the relative functionality of mouse dendritic cells, human macrophages and human T cells post
nucleofection ® . Functionality is given in percent related to non-transfected control. Functionality was analyzed by IL-6 specific
ELISA for mouse dendritic cells, TNF- a specific ELISA for human macrophages, IFN- g specific ELISA for stimulated human T cells,
and by flow cytometry using a CD25 specific antibody for both human T cell states (resting and stimulated). Experiments were
performed on a standard Nucleofector ® (mouse dendritic cells) or the 96-well Shuttle ® (human macrophages and human T cells).
excellent preservation of functionality,
tested by the ability of the transfected
cells to secret IL-6 (Figure 1) after
induction with LPS. For this purpose
immature mouse dendritic cells were incubated
with 0.1 µg/ml LPS two hours
post nucleofection ® . IL-6 secretion was
analyzed by a standard sandwich ELISA
(IL-6 ELISA MS, BioSource) and is
given in percent compared to the nontransfected
control. In addition, the
transfected cells were able to form
dendrites as shown in Figure 2.
Human macrophages
Nucleofection ® of human macrophages
revealed high efficiency (42%) combined
with low mortality as shown by
the data presented in Table 1. Functionality
of human macrophages has
been tested by measuring the secretion
of TNF- a triggered by stimulation of
the cells with 1 µg/ml LPS 24 hours
post nucleofection ® . Data shown in
Figure 1 prove the excellent conservation
of macrophage functionality post
nucleofection ® . TNF- a secretion was
analyzed by sandwich ELISA (TNF- a
96-well Shuttle ® 96-well Shuttle ®
96-well Shuttle ®
Human Macrophages /
TNF- a secretion
Resting Human T Cells /
CD 25 expression
EASIA, BioSource) and is shown in
comparison to non-transfected control
(macrophages handled as described in
the Optimized nucleofection ® Protocol
but not treated with DNA).
Human T cells
Unstimulated human T cells were
transfected according to the specific
Optimized Protocol either by using a
program suited for highest efficiency
(FI-115) or a program optimized for
highest functionality (EO-115). Stimulated
human T cells were transfected according
to the Optimized nucleofection ®
Protocol for stimulated human T cells
(transfection performance data, see
Table 1). Preservation of the biochemical
functionality of resting T cells after
nucleofection ® was analyzed by the
detection of interleukin receptor 2
(IL-2R alpha chain or CD25). Therefore,
resting human T cells were stimulated
with anti-CD3 (1 ng/µl; eBioscience) and
anti-CD28 (2 ng/µl; Research Diagnostics
Inc.) antibodies 5 hours post
nucleofection ® . For stimulated cells,
maintenance of the stimulated state
› Page 11 › www.amaxa.com
Stimulated Human T cells /
CD 25
Control Sample
96-well Shuttle ®
Stimulated Human T cells /
INF- g
was analyzed by the expression of
CD25, as well as the secretion of Interferon
gamma (IFN- g ). Human T cells
expressing the surface bound receptor
were detected with labeled anti-CD25
antibodies (PE Mouse Anti-Human
CD25; BD Pharmingen) by flow cytometry.
Secretion of IFN- g was analyzed
by a sandwich ELISA (IFN- g EASIA,
BioSource). Data (Figure 1) are given in
percent compared to non-transfected
control.
Summary
amaxa’s Nucleofector ® Technology addresses
the spectrum of cell types,
substrates and cell numbers you
encounter in your research. With more
than 50 years of combined laboratory
experience, amaxa’s team of scientists
optimizes Nucleofector ® Kits to reach
excellent transfection performance
combined with superior preservation of
cell characteristics, as proven by the
examples in this short application note
and more than 160 publications about
T cells, macrophages and dendritic cells
cited in amaxa’s citation database.
Figure 2: Mature mouse dendritic cells form dendrites
post nucleofection ® .
Immature mouse dendritic cells were transfected with
pmaxGFP using the Mouse Dendritic Cell Nucleofector ® Kit.
Two hours post nucleofection ® , cells were stimulated with LPS
to mature. Cells were analyzed 24 hours post nucleofection ®
by fluorescence microscopy for maxGFP ® expression and their
ability to form dendrites (red circle).

› Product News
Improved
Jurkat
Cells
% Transfection efficiency
Higher Efficiencies for
Jurkat Cells Than Ever Before
amaxa’s R+D Team is not only working on the development of Nucleofector ® Protocols for new cell
types, but on improving existing protocols as well. Without a doubt, Jurkat cells are among the most
frequently used blood cancer cell lines within the immunology/hematology research field. In order to
always provide products delivering the best possible results, amaxa’s R+D Team re-optimized the
existing Nucleofector ® Protocols for Jurkat E6-1 (ATCC ® TIB-152) and Jurkat ACC282 (DSMZ). These
efforts led to optimal performance for both the ATCC ® and DSMZ derived Jurkat cell lines making
these cells more transfectable than ever before.
Improved performance › Up to 35% better efficiency than before
High efficiency › Up to 88% efficiency
High viability › Up to 90% viability
High expression level › For virtually any construct
A B
100 Jurkat E6-1 (ATCC
90
80
70
60
50
40
30
20
10
0
® New protocol
TIB-152)
Former protocol 100
90
80
70
60
50
40
30
20
10
0
87% 84% 54%
X-001 X-005 C-016
High transfection efficiencies in Jurkat E6-1 (ATCC ® TIB-152) and Jurkat ACC282 (DSMZ) cell line clones.
Using pmaxGFP Jurkat E6-1 (ATCC ® TIB-152) cells were transfected with Nucleofector ® Solution V either with Nucleofector ®
Program X-01/X-001 or X-05/X-005 in comparison to the formerly recommended program C-16/C-016 (A). Nucleofector ® Program
X-01/X-001 is recommended for higher viability in Jurkat E6-1 (ATCC ® TIB-152) cells and program X-05/X-005 is recommended for
a higher expression level. Jurkat ACC282 (DSMZ) cells were transfected with Nucleofector ® Program X-01/X-001 using pmaxGFP
(B). 24 hours post nucleofection ® , cells were analyzed on a Becton Dickinson FACSCalibur for maxGFP ® expression. Cell viability
was determined as PI negative cells and ranged from 74% - 90%.
Expression level is what counts
It is widely known that different constructs are expressed differently after transfection. Vector
backbones and inserts are both responsible for this phenomenon. To make your daily work easier,
we focus not only on the transfection efficiency but also on the expression level. Therefore, the
new Nucleofector ® Protocol for the Jurkat E6-1 (ATCC ® TIB-152) recommends two different
Nucleofector ® Programs, X-01/X-001 for better viability and X-05/X-005 for a higher expression level.
The latter program guarantees the expression of virtually any construct of your interest.
› Page 12 › amaxa news # 10
% Transfection efficiency
Jurkat ACC282 (DSMZ)
74% 68%
X-001 A-017
New protocol
Former protocol

[k]
[k]
› Product News
Ordering information
Cell Line Nucleofector ® Kit V Cat. No.: VCA-1003
amaxa web information www.amaxa.com/jurkat-standard.html www.amaxa.com/improved-jurkat
› Product News
new
Expression level of maxGFP ® in Jurkat
E6-1 (ATCC ® TIB-152) cells.
Jurkat E6-1 (ATCC ® ) cells were transfected
with Nucleofector ® Solution V either with
Nucleofector ® Program X-01/X-001 or
X-05/X-005 using 2 µg pmaxGFP. 24 hours
post nucleofection ® , cells were analyzed
on a Becton Dickinson FACSCalibur for
determining maxGFP ® expression level
(X-Mean).
96-well Shuttle ® Protocols and Kits –
Human T Cells, HepG2, SH-SY5Y
› Page 13 › www.amaxa.com
4000
3500
3000
2500
2000
1500
1000
500
0
X-Mean
m › Up to 95% efficiency and excellent viability
› Low well-to-well variations
› Preserved functionality (e.g., human T cells)
› Suitable for a variety of substrates, e.g., DNA and siRNA
› Fewer cells required compared to the single cuvette Nucleofector ®
High transfection efficiency and viability in SH-SY5Y
(ATCC ® CRL-2266), HepG2 (ATCC ® HB-8065) and
stimulated human T cells with the 96-well Shuttle ® .
SH-SY5Y, HepG2 cell lines and primary stimulated human
T cells were transfected with the 96-well Shuttle ® System using
the recommended Nucleofector ® Programs and pmaxGFP.
24 hours post nucleofection ® , cells were analyzed on a Becton
Dickinson FACSCalibur with HTS options for maxGFP ®
expression. Cell viability was determined by using the
CellTiter-Glo ® Luminescent Viability Assay (Promega) or
CellTiter ® -Blue Cell Viability Assay (Promega).
100
90
80
70
60
50
40
30
20
10
0
%
X-001 X-005
Jurkat E6-1 Jurkat E6-1
(ATCC ® TIB-152) (ATCC ® TIB-152)
Other re-optimized protocols available
› COS-7 › U-937 › PC-12
Transfection efficiency
For the latest addition of 96-well Shuttle ® and regular Nucleofector ® Kits, please check our website: www.amaxa.com
Viability
HepG2 SH-SY5Y Stimulated
(ATCC ® HB-8065) (ATCC ® CRL-2266) human T cells
Ordering information 96-well Nucleofector ® Kit 96 reactions 960 reactions
Primary cells T cells, human, stimulated Human T Cell Cat. No.: VHPA-1002 Cat. No.: VHPA-2002
Cell lines SH-SY5Y Cell Line Kit SF Cat. No.: VHCA-1002 Cat. No.: VHCA-2002
HepG2 Cell Line Kit SF Cat. No.: VHCA-1002 Cat. No.: VHCA-2002

[k]
› Product News
new
Mouse and human stem cells were
transfected according to the dedicated
Optimized Protocol for nucleofection ®
using pmaxGFP.
maxGFP ® expression was analyzed 24 hours
post nucleofection ® by flow cytometry.
Transfection efficiencies given for human
stem cells are average values derived
from customer data.
Ordering information
amaxa web information
From Mouse to Human –
The Latest Nucleofector ® Kits
for Stem Cells
Adult and embryonic stem cells are perfectly suited to study cell differentiation and tissue/organ
development. The Nucleofector ® Technology presents a powerful tool to transfect these delicate cells
with high performance and preservation of functional properties. The latest additions to our stem cell
portfolio are Nucleofector ® Kits for human stem cells and a 96-well Nucleofector ® Kit for mouse
embryonic stem cells.
› Up to 90% transfection efficiency and 80% cell viability
› Excellent preservation of differentiation ability
› Suitable for a variety of substrates, e.g., DNA and siRNA
100
90
80
70
60
50
40
30
20
10
0
Transfection efficiency %
96-well 96-well
Mouse ES (E14) Mouse ES (D3) Mouse ES H9.2 H9
Mouse ES Nucleofector ® Kit 25 reactions Cat. No.: VPH-1001
96-well new Mouse ES 96-well Nucleofector ® Kit 96 reactions Cat. No.: VHPH-1001
Human CD34 + Cell Nucleofector ® Kit 25 reactions Cat. No.: VPA-1003
Human MSC (Mesenchymal Stem Cell) Nucleofector ® Kit 25 reactions Cat. No.: VPE-1001
Mouse NSC (Neural Stem Cell) Nucleofector ® Kit 25 reactions Cat. No.: VPG-1004
Rat NSC (Neural Stem Cell) Nucleofector ® Kit 25 reactions Cat. No.: VPG-1005
new Human Stem Cell Nucleofector ® Starter Kit 18 reactions Cat. No.: VPH-5002
new Human Stem Cell Nucleofector ® Kit 1 25 reactions Cat. No.: VPH-5012
new Human Stem Cell Nucleofector ® Kit 2 25 reactions Cat. No.: VPH-5022
› Page 14 › amaxa news # 10
For further information, please contact your local representative or visit www.amaxa.com

[k]
[k]
› Product News
new
Ordering information
Further expression vectors
pmaxCloning Vector –
Strong Expression of Your
Individual cDNA
Do you have difficulties in expressing your specific gene-of-interest?
One crucial aspect for obtaining high expression levels is the selection of an appropriate expression
vector. amaxa’s novel pmaxCloning vector promotes strong, constitutive expression of cloned DNA
inserts in mammalian cells. It provides gene expression under the control of a CMV promoter* and
contains a multiple cloning site for cloning purposes. With amaxa’s pmaxCloning and pmaxFP ®
vectors for fluorescent protein expression you can choose from a broad variety of expression
plasmids for your specific experimental setup.
*The CMV promoter is covered under the U.S. patents 5,168,062 and 5,385,839 and its use is permitted for research purposes only. Any other use of the
CMV promoter requires a license from the University of Iowa Research Foundation, 214 Technology Innovation Center, Iowa City, IA.
Benefit from:
› High expression rates in mammalian cells
› Multiple cloning site for convenient insertion of your gene-of-interest
› Proven performance (same backbone as positive control vector pmaxGFP ® )
Cat. No.: VDC-1040 pmaxCloning
Kan r
pmaxCloning,
2.9 kb
pUC ori
MCS Kpnl Pme I EcoR I EcoR V BamH I Nhe I Pme I Sac I
947
pmaxFP ® -Green-C Cat. No.: VDF-1011
pmaxFP ® -Green-N Cat. No.: VDF-1012
pmaxFP ® -Green-PRL Cat. No.: VDF-1013
P
PCMV
IE
SV40
poly A
chimeric
intron
GG.TAC.CGC.CAT.CAT.GAA.GTT.TAA.ACA.AGC.TTG.AAT.TCT.CTA.GAG.ATA.TCC.TGC.AGA.GAT.CTG.GAT.CCC.TCG.AGG.CTA.GCG.CGG.CCG.CGT.TTA.AAC.AGA.GCT.C
Hind III Xbal Pst I Xho I Not I
pmaxFP ® -Yellow- C Cat. No.: VDF-1021
pmaxFP ® -Yellow-N Cat. No.: VDF-1022
pmaxFP ® -Yellow-PRL Cat. No.: VDF-1023
amaxa web information www.amaxa.com/expression-vectors.html
MCS
› Page 15 › www.amaxa.com
pmaxFP ® -Red-C Cat. No.: VDF-1031
pmaxFP ® -Red-N Cat. No.: VDF-1032
pmaxFP ® -Red-PRL Cat. No.: VDF-1033

Morpholino
› Application Note
Achieving Efficient
Delivery of Morpholino Oligos
with Nucleofection ®
Shannon T. Knuth, Jon D. Moulton and Paul A. Morcos, Biology and Customer Support, Gene Tools,
LLC, Philomath, Oregon, USA
Morpholino oligos provide a stable, specific and effective antisense activity. Developmental biologists have long since
used these properties by microinjecting or electroporating Morpholinos into embryos to allow for splice-blocking or
gene knockdown. However, efficient delivery of Morpholinos in mammalian cell cultures can be a challenge. While
complex formation with lipid-based reagents is ineffective due to the non-ionic Morpholino backbone, we show here
that nucleofection ® can be used as an efficient delivery method. As such, nucleofection ® of Morpholinos offers a
straightforward alternative to siRNA-mediated gene knockdown and miRNA maturation inhibition.
RNA
DNA
Figure 1: Comparison of the chemical structure of
two-mers of Morpholino, RNA and DNA.
Two significant differences in the Morpholino backbone
compared to RNA and DNA are the non-charged phosphodiamiate
linkage between the subunits and replacement
of the five-membered sugar ring with the six-membered
Morpholine ring.
Introduction
Morpholino oligos (MOs), also known as
phosphodiamidate Morpholino oligos,
are designed to bind to complementary
RNA sequence and sterically inhibit
proteins from binding the RNA.
Morpholinos are commonly used to
knock down gene expression and alter
mRNA splicing in a variety of organisms,
tissues or cells (for a searchable database
of references see http://www.genetools.
com/Publications/). For gene
knockdowns the MOs are designed to
complement mRNA sequence in the 5’
untranslated region and/or the first
25 bases of mRNA coding sequence,
sterically inhibiting the movement
of the translation initiation complex
toward the start codon. This prevents
the large subunit of the ribosome
from assembling and, as a consequence,
causing reduced gene expression.
Morpholinos are also used to modify
pre-mRNA splicing by targeting the
oligo to a splice junction or regulatory
Advantages of Morpholinos over DNA and RNA-based gene knockdown reagents:
› Very robust and stable molecules 10, 11 due to the non-biological, chemically stable backbone structure (Figure 1)
› Exquisite specificity: at low concentrations a Morpholino with just five mispairs along its length will have
no antisense activity 12
› Largely free of non-antisense effects as evidenced by rescue of the Morpholino phenotype via injection of the
corresponding mRNA 13
› Backbone is non-toxic
› Predictable targeting
› Steric block mechanism allows for additional applications such as altering mRNA splicing and blocking microRNA maturation
› Page 16 › amaxa news # 10
site, sterically blocking binding of
snRNPs or other splice factors 1 . Spliceblocking
Morpholinos have great promise
for therapeutic use correcting and/or
reducing the impact of the mutations
that lead to b -Thalessemia 2 , Duchenne
muscular dystrophy 3 and other diseases
caused by mis-splicing 4,5 . Morpholinos
have also been used to block microRNA
maturation 6 and microRNA targets 7 ,
block ribozyme activity 8 and induce
frameshifts 9 .
However, even more so than RNA and
DNA based oligos, Morpholinos are
challenging to deliver into mammalian
cells in culture. Standard lipid-based
nucleic acid transfection reagents rely
on an ionic interaction with a charged
backbone and are not effective with
the uncharged MO backbone. While
microinjection is suitable for delivery
into organs (embryos or brains), most
commonly used transfection methods
for cultured cells are scrape loading 16 ,

› Application Note
Figure 2: Dose-dependent delivery of
Morpholinos into ON 705 HeLa cells.
Cells were transfected with indicated
concentrations of splice-correcting MO.
In the ON 705 HeLa cell line, the
Morpholino corrects a splicing mutation
splicing out a stop codon and putting
luciferase in frame. After 24 hours, the
cells were lysed and luciferase activity
and protein amount were determined.
Delivery success is proportional to the
light output (normalized to total protein
amount).
special delivery 17 , conjugated cell-penetrating
peptides 19 , Endo-Porter delivery 18
or electroporation 14 . Here, we present,
the results of our study using the
Nucleofector ® Technology for delivering
Morpholinos.
Materials and Methods
The ON 705 HeLa cell line was grown to
75-80% confluency in DMEM/F-12 media
(Invitrogen) with 10% FBS (Invitrogen).
On the day of the experiment the cells
were treated with 0.5% trypsin (Sigma),
removed from the plate and spun down.
The supernatant was removed and the
cells resuspended in 100 µl Nucleofector ®
Solution R per manufacturer’s, direction.
The indicated amount of MO was added
to each sample and mixed by vortexing.
The sample was transferred to a
nucleofection ® cuvette and transfected
using Nucleofector ® Program I-013. After
nucleofection ® , 500 µL of serum free
RPMI medium was added to each
cuvette followed by transfer of contents
to a 6-well plate. In the ON 705 HeLa
cell line, the delivery of the Morpholino
corrects a splicing mutation, splicing
out a stop codon and putting luciferase
in frame 15 . After 24 hours, the cells were
lysed and luciferase (Promega) and pro-
Figure 3: Modifications of 3' end do
not alter delivery efficiency.
Cells were transfected with 0.2 nmol
(2 µM) of a standard MO, a carboxyfluorescein
(Fl) or a lissamine (Li) labeled
MO. 24 hours post nucleofection ® , delivery
efficiency was analyzed by activity
of splice-corrected luciferase (normalized
to total protein amount).
tein (Bio-Rad) assays were carried out in
duplicate for each sample. Delivery success
is proportional to the light output
and the data is normalized per protein.
Results/Discussion
Morpholinos were delivered uniformly
in nearly 100% of the cells when imaged
by fluorescence microscopy (data not
shown). To further analyze delivery
efficiency we used our established
splice correction assay with a luciferase
reporter stably expressed in ON 705
HeLa cells. The luciferase activity
produced at the 1-8 µM range of MO
reveals a consistent increase in MO
activity (Figure 2). Generally, as shown
by the efficacy of cytosolic microinjection
of splice-modifying Morpholinos
into embryos, delivery to the cytosol is
sufficient to give nuclear splice-correcting
activity of Morpholinos. However,
average luciferase activities (Figure 2)
seem to be slightly higher with
nucleofection ® compared to activities
commonly achieved with Endo-Porter.
Effective MO amounts needed for
nucleofection ® seem to be lower than
those published for classical electroporation
14 . This suggests that nucleofection ®
could be advantageous in terms of
› Page 17 › www.amaxa.com
1600
1400
1200
1000
800
600
400
200
0
700
600
500
400
300
200
100
0
Luciferase activity
0 1 2 4 8
MO concentration (µM)
Luciferase activity
effective amounts due to its direct
nuclear delivery or to smaller reaction
volumes (higher concentrated cell
suspension).
In an additional experiment (Figure 3),
the MO concentration was fixed at 2 µM
and we compared delivery of Morpholinos
with different 3’ end modifications. The
carboxyfluorescein (Fl) has two negative
charges at physiological pH while the
lissamine (Li) (sulforhodamine B) is a
zwitterion with no net charge. We found
that there were no significant differences
in delivery of the MOs with 3’ end groups
compared to MOs with unmodified ends.
In our lab, nucleofection ® has also been
used effectively in C2C12 muscle cells
to produce a quantitative shift in the
splicing of dystrophin.
In conclusion, nucleofection ® has been
shown to allow efficient delivery of
Morpholino oligos in cultured mammalian
cells. Thus, by using nucleofection ®
for delivery, Morpholinos can now be
explored in difficult-to-transfect cell
types and primary cells as potent alternative
for other DNA- or RNA-based
gene knockdown substrates.
Std. control Fl. std. control Li. std. control
MO concentration (µM)
References
1. Morcos (2007)
Biochem Biophys Res
Commun 358(2): 521-7.
2. Kole et al. (2004)
Oligonucleotides
14(1): 65-74.
3. Yokota et al. (2007)
Expert Opin Biol Ther
7(6): 831-42.
4. Scaffidi and Misteli
(2006) Science
312(5776): 1059-63.
5. Ugarte et al. (2007)
Am J Hum Genet 81(6).
6. Kloosterman et al.
(2007) PLoS Biol
5(8): e203.
7. Choi et al. (2007)
Science 318: 271-4.
8. Yen et al. (2004)
Nature 431(7007): 471-6.
9. Howard et al. (2004)
RNA 10(10): 1653-61.
10. Hudziak et al. (1996)
Antisense Nucleic Acid
Drug Dev 6(4): 267-72.
11. Youngblood et al.
(2007) Bioconjug Chem
18(1): 50-60.
12. Summerton (1999)
Biochim Biophys Acta
1489(1): 141-58.
13. Heasman et al. (2000)
Dev Biol 222(1): 124-34.
14. Matter and Konig
(2005) Nucleic Acids
Res 33(4): e41.
15. Schmajuk et al. (1999)
J Biol Chem
274(31): 21783-9.
16. Partridge et al. (1996)
Antisense Nucleic Acid
Drug Dev 6(3): 169-75.
17. Morcos (2001) Genesis
30(3): 94-102.
18. Summerton (2005)
Ann N Y Acad Sci
1058: 62-75.
19. Wu et al. (2007)
Nucleic Acids Res
35(15): 5182-91.

› amaxa Insights
amaxa’s QA
and Production Team
As a technology company, amaxa has a duty to ensure its products
are manufactured and tested to the highest possible specifications.
The amaxa culture of continuous improvement makes this
a yet more demanding task. Each new technology must be
thoroughly evaluated and then seamlessly integrated with existing
systems in order to ensure our customers receive high quality
products that meet both their current and future needs.
Production and Quality Assurance (QA) departments are responsible
for this overall process within amaxa and project leaders
from both departments work closely with colleagues in Sales and
Marketing, Finance, Purchasing, and Scientific Support, as well as
with external partners such as suppliers of raw materials. The
Production and QA departments support Sales Managers by
reserving requested production lots for key customers or providing
lot-specific Certificates of Analysis to customers with stringent
internal validation and audit procedures.
Both the Production and QA departments at amaxa have been
undergoing some exciting changes in the last 12 months. Our
Production department has fully automated Nucleofector ®
Solutions filling due to the increasing demand for Nucleofector ®
and 96-well Shuttle ® Kits and to proactively improve both the
› Page 18 › amaxa news # 10
Dr. Christoph Zander
Head of Production
consistency and quality of amaxa Nucleofector ®
Solutions. A recently installed automation line
consists of 11 fully integrated instruments controlled
centrally by computer. The system includes
a filling carousel, in-line sealing and labeling, and
an instrument to QA test the amount of solution
filled into each bottle gravimetrically.
Dr. Christoph Zander, Head of Production, describes
the integration project as a “very challenging,
but rewarding process”. The entire project – “from
concept through to daily operation of the device” –
has taken 12 months. But Christoph is confident
that the project is not only necessary, but will also
deliver sustained improvements to customers.
“The cost of such a project is significant, but this
is a long-term investment in higher quality. Right
now, we are making new expiry-date testing
for Nucleofector ® Solutions filled with the automated
process that shows a longer shelf-life
due to more reproducible sealing of bottles. In
addition, the people who were working on the
Nucleofector ® Solution filling line now have more
time for further projects. So we are delivering
continuous improvement to customers and also
to our employees!”

› amaxa Insights
Dr. Rainer Hammermann
Head of QA
The amaxa QA
and Production Team
The Scientific Support department will be involved in monitoring
and following up with statistics to show improvements made by
the new production process and Dr. Rainer Hammermann, Head of
QA, ensures that each piece of customer feedback is recorded and
that each individual complaint is followed up to the customer’s
satisfaction. “Scientific Support is a very important channel for
monitoring both emerging customer needs and also customers’
perception and acceptance of amaxa quality standards. We are not
standing still – instead we are anticipating our customers needs for
the next five years and beginning projects that will help to further
associate the name amaxa with high quality and dependable
products.”
amaxa cGMP Kits (current Good Manufacturing Practice) and their
components are manufactured and filled under Grade A clean
room conditions (EU Guide to Good Manufacturing Practice-
Manufacture of Sterile Medicinal Products, 1997), meaning that
air in the manufacturing facility is monitored and that each
cubic meter of air contains not more than 3500 particles of
0.5 µm diameter or larger. Manufacturing each kit component
in full compliance with cGMP regulations means that these kits are
suitable for use in disease research and development of
pharmaceuticals. Scientists working in heavily regulated areas
such as protein production and drug discovery can now use amaxa
› Page 19 › www.amaxa.com
cGMP Kits for their work without first validating
kit performance and other features. In contrast
to other manufacturers, amaxa includes a
more detailed and batch-specific Certificate of
Analysis (CoA) into each cGMP Nucleofector ® Kit,
providing an ‘audit trail’ of performance and
QA test data for each batch of kits. Rainer believes
that this demonstrates “full disclosure of
data in order to ensure reliable research results,
thus protecting the safety of researchers in
development departments of the pharmaceutical
industry.”
As the needs of Nucleofector ® users evolve and
expand into new application areas such as stem
cell research and drug discovery, amaxa Production
and Quality Assurance will continue to set
industry standards and enable researchers to be
increasingly confident in their research data.

› Hot Topic
Achieve Your Research Objectives
with amaxa Transfection Services
All amaxa services are designed to meet your needs and to assist you in successfully achieving
your transfection goals. amaxa’s team of scientists have years of transfection experience and will
provide you with fast, reliable results along with expert scientific customer support.
Numerous pharmaceutical and biotech companies, in both the US and Europe, have already worked
with amaxa to generate stable clones and optimize transfection conditions for their
Nucleofector ® II or 96-well Shuttle ® Devices.
These projects include the generation of stable cell clones expressing drug targets and the
generation of reporter cells for several signal transduction pathways. Moreover amaxa has
extensive experience in working with more than 100 different cell lines and primary cells and has
successfully created stably transfected clones from cells that are very difficult-to-transfect, like
J774, Jurkat, HL-60, BJAB, MCF-7, and suspension CHOs.
amaxa scientists will optimize transfection protocols using our unique non-viral technology for
any cell type and substrate, allowing you the freedom to focus your expertise on analysis and
experimentation, not the creation of the clone or protocol.
amaxa's results and expertise are proven: Nucleofection ® is used in expression studies, RNAi
knockdown experiments, transient protein expression, target screening, target validation, and
stable clone generation. It is deployed on the lab bench and on high-throughput automated
platforms. More than 4000 industrial-based scientists are now using nucleofection ® to help
achieve their business objectives.
amaxa web information www.amaxa/salesreps
› Page 20 › amaxa news # 10
”Thanks again for your excellent support,
it has been a very pleasant experience
to work together with you and
I will be glad to further recommend
your service for assay development!“
Dr. Helmut Glantschnig
Senior Research Biochemist, US Pharmaceutical Company
Tell us how we can help you achieve your objectives,
or find out more about our capabilities by contacting
our dedicated sales team.

› FAQ
FAQs on Nucleofection ® and
Preservation of Functionality
› Are the rat and mouse hepatocytes still
functional after nucleofection ® ?
›› Yes, transfected mouse hepatocytes show
albumin synthesis rates comparable to those
of untransfected cells. Moreover, the morphology
and polarization of rat hepatocytes is not
affected by nucleofection ® .
› Are there any effects on the differentiation
of neural stem cells following nucleofection ® ?
›› The ability of transfected rat or mouse neural
stem cells to be subsequently differentiated
into a variety of cell types is well documented
in scientific literature, for example:
› Nucleofection ® of neural stem cells (NSC)
does not affect their functionality or impinge
on their ability to be subsequently differentiated
into oligodendrocytes [Copray et al.
(2006) Stem Cells 24(4): 1001-1010].
› Have you analyzed whether nucleofection ®
influences differentiation in mouse ES cells?
›› During the optimization process of a
Nucleofector ® Protocol, great care is taken
about preservation of functionality of the
cells post transfection. Internal experiments
showed that nucleofection ® does not alter the
differentiation ability of mouse ES. This was
investigated by analyzing the expression level
of the transcription factor GATA-6. Results
have also shown that the differentiation process
itself is not triggered by nucleofection ® .
This is well documented in literature showing
unaltered differentiation potential of mouse
ES cells, e.g. (Lakshmipathy et al., Stem Cells
22:531-543).
› Page 21 › www.amaxa.com
› Have you tested whether transfected macrophages
maintain functionality?
›› Yes, we analyzed the secretion of TNF-a triggered by stimulation
of the cells with LPS 24 hours post nucleofection ® . In comparison
to the untransfected control, the functionality is 70%.
› Is it possible to stimulate transfected T cells?
›› Yes, transfected resting T cells can be stimulated post
nucleofection ® . It is important to wait at least 4 hours before
stimulation (e.g., incubation in Human T Cell Medium provided
by amaxa). Stimulating immediately after nucleofection ® may
lead to increased cell mortality and poor activation of cells.
For optimal stimulation of the transfected cells, we recommend
coating 96-well plates for stimulation with a solution of
anti-CD3 antibody [OKt 3; eBioscience, Cat. No. 14-0037-82]
and anti-CD28 antibody [15E8; Research Diagnostics Inc.,
Cat. No. RD1-111650clb]. For stimulated cells, maintenance of the
stimulated state was analyzed by expression of CD25 as well as
the secretion of interferon gamma.
› Is it possible to transfect mRNA of tumor cells into
antigen-presenting cells like B cells or dendritic cells
with the Nucleofector ® Technology?
›› Yes. Recently Coughlin and co-workers showed that RNA
transfected CD40-activated B cells or DCs induce functional
T cell responses against viral and tumor antigen targets
(Coughlin et al. (2004) Blood 103:2046-2054).
In short, the authors found:
› mRNA uptake in B cells and dendritic cells up to 80%.
› CD40-activated B cells or dendritic cells induce functional
T cell responses against viral and tumor antigen targets.
› mRNA transfection is less toxic and gave higher efficiencies.
amaxa web information www.amaxa.com/faq

› amaxa Worldwide
Distributor Overview
Australia I Quantum Scientific Pty Ltd I 1/31 Archimedes Place I Murarrie, QL 4172, Australia
New Zealand Phone +61-1800-777 168 I Fax +61-1800-625 547 I rods@quantum-scientific.com.au I www.quantum-scientific.com.au
Canada ESBE Scientific I 80 McPherson St. I Markham ON L3R 3V6, Canada
Phone +1-905-475-8232 +1-800-268-3477 I Fax +1-905-475-5688 I custserv@esbe.com I www.esbe.com
China/Hongkong Jingmei Biotech co. Ltd. I 14 FI, Kaiyuan Plaza I No. 7001 Beihuan Ave. I Shenzhen 518034, China
Phone +86-7558-3546 194 I Fax +86-7558-3546 196 I shenzhen@jingmei.com I www.jingmei.com
Croatia INEL-medical technics I Avenija Dubrovnik 10A I 10 000 Zagreb, Croatia
Phone +385-1-6520 546/556 I Fax +385-1-6520 966 I info@inel-mt.hr I www.inel-mt.hr
Czech Republic Central European Biosystems Ltd. I U Habrovky 247/11 I 140 00 Prague 4, Czech Republic
Phone +420-603-533-444 I Fax +420-257-013-411 I cebiosys@cebiosys.com I www.cebiosys.com
Denmark I REACTIONLAB AS I Vassingerödvej 91C I 3540 Lynge, Denmark
Iceland Phone +45-7027 9595 I Fax +45-7027 9596 I info.denmark@reaction-lab.com I www.reaction-lab.com
Finland REACTIONLAB OY I Tykistökatu 4 D I FIN-20520 Turku, Finland
Phone +358-2-410 11 46 I Fax +358-2-410 11 23 I info.finland@reaction-lab.com I www.reaction-lab.com
Greece BioAnalytica S.A. I 3A-5 Ilission Street I 11528 Athens, Greece
Phone +30-210-640 03 18 I Fax +30-210-646 27 48 I bioanalyt@bioanalytica.gr I www.bioanalytica.gr
Hungary Central European Biosystems Kft. I 2040 Budaörs I Gyár u.2 Innovációs Központ, Hungary
Phone +36-23 502 195 I Fax +36-23 502 196 I cebiosys@cebiosys.com I www.cebiosys.com
India Genetix Biotech Asia Pvt. Ltd. I 71/1, Najafgarh Road I Near Moti Nagar I New Dehli - 110015, India
Phone +91-11-4142-7031 I Fax +91-11-25419631 I genetix@genetixbiotech.com
Israel Rhenium Co. LTD I P.O.B. 3580 I Jerusalem 91035, Israel
Phone +972-2-5335599 I Fax +972-2-533559 I dana@rhenium.co.il I www.rhenium.co.il
Italy Instrumentation Laboratory spa I Viale Monza 338 I 20128 Milano, Italy
Phone +39-0225-22 766 I Fax +39-0225-22 736 I beretta@itmil.ilww.com I www.ilww.com
Japan Wako Pure Chemical I Industries, Ltd. I 1-2, 3 Doshomach, Chuo-ku I Osaka 540-8605, Japan
Phone +81-6-6203-2756 I Fax +81-6-6201-5965 I kushida.katsutoshi@wako-chem.co.jp I www.wako-chem.co.jp
Korea Young-Wha Scientific Co., Ltd. I Gujung Bldg. 577-5 I Shinsa-dong, Gangnam-gu I Seoul 135-891, Korea
Phone +82-2-2140-5484 I Fax +82-2-2140-5405 I ycjoo@youngwha.com I www.youngwha.com
Malaysia I Research Biolabs I 73 Ayer Rajah Crescent #04-05 I Ayer Rajah Industrial Estate I Singapore 139952
Indonesia Phone +65 6777 5366 I Fax +65 6778 5177 I sales@researchbiolabs.com I www.researchbiolabs.com
Mexico Quimica Valaner I Jalapa 77 Col. Roma I Mexico D.F. 06700
Phone +52-5525 5725 I Fax +52-5525 5625 I servicioaclientes@valaner.com I www.valaner.com
Singapore Research Biolabs Pte Ld I 73 Ayer Rajah Crescent #04-05 I Ayer Rajah Industrial Estate I Singapore 139952
Phone +65 6777 5366 I Fax +65 6778 5177 I sales@researchbiolabs.com I www.researchbiolabs.com
Slovakia Central European Biosystems s.r.o. I Kazanská 12 I 84102 Bratislava, Slovakia
Phone +421-905 534 365 I Fax +421-245 640 437 I cebiosys@cebiosys.com I www.cebiosys.com
Slovenia MEDILINE d.o.o. I Perovo 30, P.O. Box 5 I SI-1241 Kamnik, Slovenia
Phone +386-01-830 80 40 I Fax +386-01-830 80 70 I info@mediline.si I www.mediline.si
South Africa Celtic Diagnostics I PO Box 13674 I Mowbray 7705 I South Africa
Phone +27 21 685 1112 I Fax +27 21 685 1117 I Dermot@celticdiagnostics.com I www.celticdiagnostics.com
Spain I Portugal IZASA, S.A. I Aragón 90 I 08015 Barcelona, Spain
Phone +34-902-20 30 90 I Fax +34-902-22 33 66 I CLC@izasa.es I www.izasa.es
Sweden I Norway REACTIONLAB SVERIGE AB I Box 16023 I 750 16 Uppsala, Sweden
Phone +46-18-14 90 00 I Fax +46-18-14 90 10 I info.sweden@reaction-lab.com
Latvia I Lithuania
Estonia
Taiwan Taigen Bioscience Corporation I 3F, No. 306, Section 4 I Chen-Der Road I Taipei, Taiwan
Phone +886-2-2880 2913 I Fax +886-2-2880 2916 I tech@taigen.com I www.taigen.com
Turkey ATC Saglik Ltd. I 7. Cadde, 73/A I 06610-Birlik Mahallesi I Cankaya, Ankara, Turkey
Phone +90-312-496-43-19/20 I Fax +90-312-496-43-18 I erkurt@atcmed.com I www.atcmed.com
› Page 22 › amaxa news # 10

› amaxa Insights
We look forward to welcoming
you at our booth at any of
these meetings and shows!
Meet amaxa in 2008:
Feb. 10 - 13 ARABLAB the Expo 2008
Dubai, United Arab Emirates, booth #468, www.arablab.com
Mar. 13 - 14 RNAi2008
Oxford, England
www.libpubmedia.co.uk/Conferences/RNAi2008/Home.htm
Apr. 1 - 4 Analytica
Munich, Germany, booth #211, www.analytica-world.com
Apr. 5 - 9 Experimental Biology
San Diego, California, USA, booth #312, www.eb2008.org
Apr. 12 - 16 AACR – Annual Meeting of the American Association
for Cancer Research
San Diego, California, USA, www.aacr.org
Jun. 11 - 14 6th ISSCR Annual Meeting
Philadelphia, Pennsylvania, USA, www.isscr.org
Jul. 5 - 8 EACR20
Lyon, France, booth #D3, www.fecs.be
› Page 23 › www.amaxa.com
Nov. 15 - 19 SFN – Annual Meeting of the Society for Neuroscience
Washington DC, USA, www.sfn.org
Nov. 18 - 20 BSI – Annual Meeting of the British Society for Immunology
Glasgow, Scotland, www.immunology.org/congress/2008/index.php
Dec. 13 - 17 ASCB – Annual Meeting of the American Society
for Cell biology
San Francisco, California, USA www.ascb.org
www.amaxa.com/meetings

WAA-1001_10
amaxa AG
Nattermannallee 1
50829 Koeln
Germany
amaxa Inc.
205 Perry Parkway
Suite 7
Gaithersburg, MD 20877
USA
webinars
2008
We hope
you’ll join us!
WEBINAR 5
WEBINAR 6
WEBINAR 7
Scientific Support
Europe/World
Phone +49(0)221-99199-400
Fax +49(0)221-99199-499
e-mail scientific-support@amaxa.com
Scientific Support
USA
Phone (888) 632-9110
Fax (888) 632-9112
e-mail scientific-support.US@amaxa.com
Ordering
Europe/World
Phone +49(0)221-99199-500
Fax +49(0)221-99199-599
e-mail order@amaxa.com
Ordering
USA
Phone (240) 632-9110
Fax (888) 632-9112
e-mail order.US@amaxa.com
We are pleased to announce
the schedule for the 2008 webinar series:
Cell Line Optimization and Basic Nucleofector ® Kits
› Tuesday, March 18, 2008
9:30 A.M. CET (Europe) / 5:30 P.M. JST (Japan)
› Wednesday, March 19, 2008
2:00 P.M. EST (US) / 11:00 A.M. PST (US)
Nucleofection ® of Stem Cells
› Tuesday, June 3, 2008
10:00 A.M. CET (Europe) / 5:00 P.M. JST (Japan)
› Wednesday, June 4, 2008
2:00 P.M. EST (US) / 11:00 A.M. PST (US)
Improving Nucleofection ® Experiments
› Tuesday, September 16, 2008
10:00 A.M. CET (Europe) / 5:00 P.M. JST (Japan)
› Wednesday, September 17, 2008
2:00 P.M. EST (US) / 11:00 A.M. PST (US)
Registration now available at: www.amaxa.com/webinars