Frank Narz, Silke Janhsen, and Ute Krger
QIAGEN GmbH, Hilden, Germany
Primary neurons are extremely difficult to transfect, making genetic modification
of neuronal cells by delivery of either siRNA or plasmid DNA challenging
). Recently, Krichevsky and Kosik reported silencing
of endogenous genes in primary rat hippocampal and cortical neurons using
QIAGEN siRNA and TransMessenger
In this study, we investigated whether TransMessenger Reagent (originally
developed for RNA transfection of eukaryotic cells) would provide efficient
transfection of DNA. We generated cultures of primary cortical neurons using
CryoCells from rat brain cortex (QBM Cell Science, www.qbmcellscience.com
which contain a mixture of neuronal and glial cells. The cultured cells
expressed characteristic markers and showed typical morphology and differentiation.
We transfected the cells with two plasmids, one encoding green fluorescent
protein (GFP) and the other encoding -galactosidase (-gal).
No obvious signs of toxicity were detected after transfection. The combination
of TransMessenger Reagent and CryoCells allowed efficient transfection of
DNA into primary cortical neurons.
Materials and methods
CryoCells, Rat Brain Cortex (QBM Cell Science) were cultured according to
the manufacturer's instructions. Briefly, to start a culture, a vial of
CryoCells (containing 4 x 106 viable cells before freezing) was thawed and
the cells were plated in Neurobasal Medium with B27 Supplement
96-well plates at a density of 120,000 cells/well in a volume of 200 l/well.
Cells were transfected in a 96-well format on the seventh day after plating.
The indicated amounts of DNA (0.1-0.5 g/well of a GFP- or -gal-encoding
plasmid) were condensed for 5 minutes with Enhancer R and Buffer EC-R in
a total volume of 30 l. The ratio of DNA to Enhancer R was 1:4. TransMessenger
Reagent (1-4 l) was added in a total volume of 20 l Buffer
EC-R and complex formation proceeded for 10 minutes. 100 l Neurobasal/B27
medium was added to the transfection complexes, medium was removed from
the cells, and the complexes were transferred to the cells. After 6 hours
of incubation, the complexes were removed and fresh medium was added.
For immunofluorescence, cells were fixed using 4% paraformaldehyde 24 hours
after transfection. Immunofluorescence was performed according to standard
procedures using primary antibodies directed against MAP2 (a neuron-specific
marker protein) and GFAP (an astrocyte-specific marker protein) and Cy3-labeled
-galactosidase activity was measured 48 hours after transfection.
Cells were lysed and enzyme activity was quantified by a colorimetric assay
using ONPG and a -galactosidase standard curve.
Results and discussion
To optimize the transfection protocol, transfections were performed
using different amounts of plasmid DNA and TransMessenger Reagent. In each
well of a 96-well plate, 0.1, 0.3, or 0.5 g plasmid DNA were used,
in combination with 1-4 l of TransMessenger Reagent. Six replicates
were set up for each combination.
Transfection efficiencies were quantified by measurin
activity in crude cell lysates of the cultures 48 hours after transfection
"). Increasing the
amount of DNA from 0.1 g per well to 0.3 g per well resulted
in higher transfection efficiency, but using 0.5 g DNA per well did
not provide a significant further increase. The amount of TransMessenger
Reagent used also affected the efficiency. These results show that amounts
of DNA and TransMessenger Reagent should be optimized.
Activities of -galactosidase 48 hours
after transfection. Cells were transfected in a 96-well format using the
indicated amounts of DNA and TransMessenger Reagent.
However, analyzing transfection efficiency using
a -gal assay did not differentiate between transfected neuronal cells
and transfected astrocytes. To estimate the proportion of transfected neuronal
cells, cells were transfected with an expression vector encoding GFP. "Low
Toxicity Using TransMessenger Reagent
" shows live cells 24 hours
after transfection and untransfected control cells. In the bright-field
images, there are no obvious signs of toxicity in the transfected cells.
GFP fluorescence is very bright throughout the transfected neurons and even
thin neurites are clearly visible. The neurite network is intact and the
neurons are well differentiated, showing that transfected neurons maintain
their characteristic morphology.
Low Toxicity Using TransMessenger Reagent
A: Bright-field image of untransfected control
cells; B: bright-field image of live cells 24 hours
after transfection of
a GFP-encoding plasmid; C: fluorescence image of live cells 24 hours after
transfection (same visual field as B). Cells were transfected in a 96-well
format using 0.3 g of DNA and 2 l of TransMessenger Reagent
To distinguish the cell types in the CryoCell cultures, cell-specific markers
were detected using monoclonal primary antibodies (anti-MAP2 antibodies
for neurons, anti-GFAP antibodies for astrocytes, the major subtype of glial
cells in the brain). "Excellent Morphology of
" shows a typical result. Staining with anti-MAP2
antibodies reveals a well-developed neurite network and brightly stained
somata, indicating good morphology and differentiation of the neurons. Staining
with anti-GFAP antibodies shows glial-cell astrocytes with a stellate morphology.
Serum-free cultures contain many fewer astrocytes than serum-containing
cultures (data not shown). Transfected and untransfected cultures display
the same GFAP- and MAP2-staining pattern. The immunofluorescence results
show that primary cultures of cortical neurons generated from CryoCells
have morphologies and characteristics comparable to cultures derived from
freshly prepared cells.
Excellent Morphology of CryoCell Cultures
Cells stained with anti-MAP2 primary antibodies
and Cy3-labeled secondary antibodies, 24 hours after transfection: A: Untransfected
cells; B: transfected cells, anti-MAP2 staining; C: transfected cells, GFP
fluorescence; D: superimposed images from B and C. Cells stained with anti-GFAP
primary antibodies and Cy3-labeled secondary antibodies: E: Untransfected
cells; F: transfected cells, anti-GFAP s
taining; G: transfected cells, GFP
fluorescence; H: superimposed images from F and G. Cells were transfected
in a 96-well format with 0.3 g DNA and 2 l TransMessenger
Reagent per well.
The superimposed images in Figure "Excellent
Morphology of CryoCell Cultures"
show that the cells expressing
GFP are positive for MAP2 and negative for GFAP and are therefore neurons.
In this typical visual field, approximately 10% of the neurons are transfected.
In general, the efficiency of transfection in cultured CryoCells was 5-10%.
This is a high efficiency for neurons, which are notoriously difficult to
transfect. The transfected neurons are again well differentiated and GFP
fluorescence is seen in their neurite network and somata. "Excellent
Morphology at Higher Magnification
" illustrates the excellent morphology
of transfected neurons.
Excellent Morphology at Higher Magnification
Cortical neurons 24 hours after transfection
with a plasmid encoding GFP. Cells were stained with anti-MAP2 primary antibodies
and Cy3-labeled secondary antibodies. A: Anti-MAP2 staining; B: GFP fluorescence;
C: superimposed images. Cells were transfected in a 96-well format using
0.3 g of DNA and 2 l of TransMessenger Reagent per well.
- Although primarily recommended for transfection of RNA, TransMessenger Reagent
can be used to efficiently transfect DNA into primary cortical neurons,
enabling genetic modification of these cells.
CryoCells from rat brain cortex can be used to generate high-quality cultures
of primary cortical rat neurons, and are a convenient and practical su
for freshly prepared cells.
- Washbourne, P. and McAllister, A.K. (2002) Techniques
for gene transfer into neurons. Curr. Opin. Neurobiol. 12, 566.
- Krichevsky, A.M. and Kosik, K.S. (2003) Efficient
RNAi-mediated gene silencing in neuronal cells using QIAGEN siRNA and
TransMessenger Transfection Reagent. QIAGEN News 2003, No. 1, 11.
- Krichevsky, A.M. and Kosik, K.S. (2003) RNAi functions
in cultured mammalian neurons. Proc. Natl. Acad. Sci. USA 99, 11,926.
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