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
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
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
). 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").
- 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.
- Krichevksy, A., and Kosik, K. (2002) RNAi functions
in cultured mammalian neurons.
- 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,
- Caceres, A., Muatinoem J., and Kosik, K. (1992) Suppression
of MAP2 in cultured cerebellar macroneurons inhibits minor neurite formation.
Neuron. 9, 607.
Reagent Selector Kit Handbook
* Data excerpted from Krichevsky, A.M. and Kosik, K.S. (2002) RNAi functions
in cultured mammalian neurons.
Source:Page: All 1 2 3 4 5 Related biology technology :1
. Efficiently Insert Unique Restriction Sites into Plasmid Vectors2
. Efficient Cleavage of Fusion Proteins to Yield Native Amino Termini3
. Mammalian Expression Vector for Efficient Cloning of PCR Fragments4
. Efficient Transfection of Neurospora Crassa5
. Efficient Transfection of Neurospora Crassa6
. Efficient Recovery of Ultrapure Plasmid DNA7
. Efficient and Reliable PCR Setup Using Eppendorf MasterMix8
. Efficient DNA transfection of primary CNS neurons using TransMessenger
. Cloning Based on Efficient Three-Fragment Assembly
. Efficient and Reliable Linear
Amplification of cRNA11
. Efficient Delivery of siRNAs to Human
Primary Cells: Electroporation
vs. Chemical Transfection