Anna M. Krichevsky and Kenneth S. Kosik
Center for Neurologic Diseases, Brigham and Womens Hospital, Harvard Medical School, Boston, MA, USA Genetic manipulations using neuronal cells are notoriously difficult. Therefore, efficient delivery of short interfering RNA (siRNA) to primary neuronal cultures is of critical importance for RNA interference (RNAi)-mediated gene suppression. Here we describe successful targeted suppression of gene expression using TransMessenger Transfection Reagent and synthetic siRNA directed against microtubule-associated protein 2 (MAP2). RNAi has become a powerful tool used to knock down the expression of genes in cultured mammalian cells. However, neuronal cells, which are difficult to manipulate, have seemed to be more resistant to RNAi than other cell types. The reasons for this are unclear. Differences in siRNA uptake across the neuronal cell membrane or the RNAi pathway itself are possible causes. In this study, synthetic siRNA was used to successfully suppress expression of MAP2.

Materials and methods Two QIAGEN siRNAs targeted against MAP2 mRNA were designed with a 5' phosphate, 3' hydroxyl, and two-base overhangs on each strand (see Table "siRNA sequences used to target endogenous MAP2 mRNA in neuronal cells"). Transfections of siRNA for gene targeting were performed using TransMessenger Transfection Reagent as described in the TransMessenger Transfection Reagent Handbook. siRNA (1 g per well) was condensed with Enhancer R and complexed with 4 l TransMessenger Reagent. The transfection complexes were diluted in 900 l of neurobasal medium and added directly to the cells. Medium containing transfection complexes was removed and replaced with neurobasal medium after 2 hours incubation. Cells were stained and analyzed after transfection. Double immunofluorescence labeling and image analysis was performed as described previously (1). siRNA sequences used to target endogenous MAP2 mRNA in neuronal cells Results and discussion The effect of siRNA-mediated RNAi on neuronal cells was tested using siRNA targeted against MAP2 endogenous mRNA. During the course of this study, it was noted that siRNA was more readily introduced into cells than plasmid DNA was. Cells were targeted with siRNA 58 days after plating. Double immunofluorescence labeling of MAP2 and the unrelated protein actin was performed 48 to 68 hours after transfection. The expression of MAP2 was specifically reduced in 7080% of cells, whereas the expression of an unrelated protein (f-actin) was unaffected (see Figure "Efficient Transfection of Primary Neuronal Cells Using QIAGEN siRNA and TransMessenger Reagent"). Transfection of either siRNA targeting MAP2 (siRNA1 or siRNA2) resulted in an identical level of inhibition of MAP2 expression. Efficient Transfection of Primary Neuronal Cells Using QIAGEN siRNA and TransMessenger Reagent Primary neuronal cells from rats were transfected with a control nonspecific siRNA or an siRNA directed against MAP2 using TransMessenger Transfection Reagent. Cells were stained 4868 hours after transfection using mouse monoclonal anti-MAP2 antibodies and Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (MAP2) and Alexa Fluor 594-conjugated phalloidin, which binds to f-actin and serves here as an expression control. Suppression of MAP2 Leads to Defective Neuronal Development Primary neuronal cells from rats were transfected with an siRNA directed against MAP2 using TransMessenger Transfection Reagent. Primary cultures were targeted with siRNA 3 hours after plating and analyzed over 2840 hours. Cells were stained using mouse monoclonal anti-MAP2 antibodies and Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (MAP2) and Alexa Fluor 594-conjugated phalloidin. The cell with silenced MAP2 expression (arrowed) exhibits severely defective filopodial elaboration.

MAP2 has been shown to be required to overcome early transitions of neuritic development and to begin elongation of neuritic processes (2, 3). Phenotypic effects of MAP2 suppression can be observed early on after plating so primary neuronal cultures were targeted with siRNA 3 hours after plating and the effect was analyzed over 28 to 40 hours. Within the shorter time frame, fewer neurons suppressed MAP2 expression, however the degree of MAP2 suppression correlated with a recognized defect in filopodia elaboration (see Figure "Suppression of MAP2 Leads to Defective Neuronal Development").

Conclusions

• Our results show that TransMessenger Transfection Reagent can be used to successfully transfect primary neuronal cells with synthetic QIAGEN siRNA and effectively suppress target gene expression. Transfection of synthetic siRNA using the non-liposomal TransMessenger Reagent is an attractive alternative to transfection using other lipid-based reagents, which are often toxic to neuronal cells.
  
  
• As primary neuronal cells are difficult to transfect with DNA, the high transfection efficiency acheived using siRNA and TransMessenger Transfection Reagent (7080%) suggests that neuronal cells, at least, are better suited to RNAi experiments using synthetic siRNA than RNAi experiments that require plasmid DNA to be transfected.
  
  
• The application of RNAi techniques to neurons, a setting where genetic manipulations have traditionally proven difficult, will be a valuable tool in studies on neuronal gene-regulation and function.

References

1. Krichevksy, A., and Kosik, K. (2002) RNAi functions in cultured mammalian neurons.
   
   
2. Proc. Natl. Acad. Sci. USA 99, 11926. Gonzalez-Billault, C. (2002) Participation of structural microtubuleassociated proteins (MAPs) in the development of neuronal polarity. J. Neurosci. Res. 67, 713.
   
   
3. Caceres, A., Muatinoem J., and Kosik, K. (1992) Suppression of MAP2 in cultured cerebellar macroneurons inhibits minor neurite formation. Neuron. 9, 607.
4. Transfection Reagent Selector Kit Handbook

* Data excerpted from Krichevsky, A.M. and Kosik, K.S. (2002) RNAi functions in cultured mammalian neurons.


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