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 (1). Recently, Krichevsky and Kosik reported silencing of endogenous genes in primary rat hippocampal and cortical neurons using QIAGEN siRNA and TransMessenger Transfection Reagent (2,3 ).

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 on poly-D-lysine-coated 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 secondary antibodies.

-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 g -galactosidase activity in crude cell lysates of the cultures 48 hours after transfection ("Efficient 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.

Efficient Transfection 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 per well.

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 CryoCell Cultures" 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.

Conclusions

• 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 bstitute for freshly prepared cells.

References

1. Washbourne, P. and McAllister, A.K. (2002) Techniques for gene transfer into neurons. Curr. Opin. Neurobiol. 12, 566.
   
2. 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.
   
3. 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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