( taining; G: transfected cells GFP ...)Efficient DNA transfection of primary CNS neurons using TransMessenger ,,, Transfection Reagent

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
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