High-efficiency transfection of short hairpin RNAs-encoding plasmids into primary hippocampal neurons.

The transfection of expression constructs encoding a variety of transgenes is a widely used method to study gene function in cultured cells. Especially when the efficiency of the knock-down of target proteins via small interfering RNAs (siRNAs) is to be determined by quantitative Western blotting, large proportions of untransfected cells compromise the analysis. Achieving high transfection efficiencies in postmitotic cells, such as neurons, poses a particular problem in that these cells cannot be selected for the expression of the transgene following transfection. It is therefore important to develop protocols that allow for the highly efficient transfection of these cells. In the present study, we identify three important parameters that prove especially useful for chronically difficult to transfect short hairpin RNA (shRNA)-encoding plasmids: the amount and quality of the plasmid DNA used and the use of new nucleofection programs. Combining those changes increases the rate of transfected cells from less than 5% to up to approximately 80%. Importantly, these high transfection efficiencies can be obtained while maintaining good cell viability and normal cellular development. Taken together, these improvements allow for a detailed biochemical and phenotypical analysis of neurons that have been nucleoporated with a wide variety of shRNAs. 2008 Wiley-Liss, Inc.