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Efficient DNA transfection of primary CNS neurons using TransMessenger ,,, Transfection Reagent

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
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