Jochen Meier, Ivonne Strmel, Radu Iosub, Sonja Schmidt,
and Rosemarie Grantyn
Developmental Physiology, Johannes Mller Institute, Humboldt University
Medical School (Charit), Berlin, Germany
Efficient delivery of DNA to primary neuronal cell cultures is of critical
importance for extending our knowledge of the molecular basis of neuronal
physiology. Here, we describe the use of Effectene Transfection Reagent
for successful transfection of primary differentiated neurons in rat superior
Enhanced green fluorescent protein (EGFP) chimeras have proven to be a powerful
tool for studying the underlying cellular mechanisms for dynamic remodeling
of synaptic compounds in living neurons (1). However, a major obstacle to
the use of such chimeras is an effective gene delivery method. Gene delivery
based on non-viral DNA vehicles is an attractive alternative because it
can be applied under a wide range of laboratory conditions. We developed
a transfection procedure using Effectene Reagent and found it highly suited
for easy and efficient transfection of differentiated neurons in primary
cultures from rat superior colliculus. Effectene Reagent shows low cytotoxicity
and delivers efficient transfection using small amounts of DNA. Furthermore,
transfection can be carried out in the presence of serum.
Materials and methods
Superior colliculus neurons were prepared from embryonic day 19 Wistar rats,
and cells were plated at a density of 7.5 x 104 cells/cm2 on laminin-coated
glass coverslips (16 mm). Cultures were maintained in neurobasal B27 medium
supplemented with 1% fetal calf serum. After neuron attachment, coversli
were transferred (cell-side down) to dishes containing a confluent layer
of collicular glial cells and were cultured for up to 3 weeks. On the second
day in vitro, cytosine arabinoside (4 M) was added to inhibit glial
cell proliferation. Transfection was performed on day 9 in vitro using various
ratios of EGFP plasmid DNA (carrying a CMV promoter and isolated from DH5alpha
E. coli using a QIAGEN Plasmid Kit) to Effectene reagent (Figure 1).
Transfection was performed as described in the Effectene Transfection Reagent
Handbook. During DNAEnhancer Effectene Reagent complex formation,
the coverslips containing the neurons were removed from the glial cell culture
and placed cell-side up in 350 l fresh, pre-warmed culture medium
in the well of a 24-well plate. Culture medium (350 l per well) was
added to transfection complexes and mixed by pipetting up and down. Diluted
transfection complexes were then added to neuronal cultures and incubated
for 4.5 hours. After incubation, the coverslips were returned to the glial
cell culture (cell-side down), and cells were cultured for 24 hours before
analysis of expression. All transfections were performed in triplicate.
Control cells were exposed to Effectene Reagent complexed with plasmid DNA
lacking a CMV eukaryotic promoter. Immunofluorescence was performed as previously
Efficient Transfection Using Effectene Reagent
Figure 1 Dependence of transfection efficiencies
on transfection conditions. Transfection efficiencies are expressed as the
percentage of neurons that are GFP-positive. Note that transfection efficiency
decreased when more than 5 l of Effectene Reagent was added. Highest
transfection efficiencies were obtained u
sing 200 ng of DNA in combination
with 25 l of Effectene Reagent per well. For culture and transfection
conditions, see Materials and methods.
Briefly, fixed and permeabilized cells were incubated for 1 hour at room
temperature with a monoclonal anti-microtubule-associated protein 2 (MAP2)
antibody. Cells were washed and incubated with a Cy3-conjugated secondary
antibody for 45 minutes at room temperature.
Results and discussion
Figure 1 shows transfection efficiency in randomly chosen view-fields, as
determined by counting the number of cell nuclei, MAP2-positive neurons,
and GFP-positive fluorescent neurons. The highest transfection efficiency
was obtained using 200 ng of DNA with 25 l of Effectene Reagent.
Under these conditions, Effectene Reagent is also suitable for transfection
of glial cells (data not shown). Use of 10 l Effectene Reagent or
exposure of cells to complexes for longer than 5 hours was toxic to neurons.
Figure 2 shows two MAP2-positive transfected neurons (green) in a culture
that was treated with 200 ng DNA and 5 l of Effectene Reagent. Nuclei
(blue) of cells not reacting with MAP2 antibodies are glial-cell nuclei.
Note that the nuclei of transfected neurons are not fragmented, which is
an indicator of good cell health. In control transfection experiments (i.e.,
when the plasmid lacking the eukaryotic promoter was used), green fluorescence
was not observed.
Transfected Primary Neuronal Cells
Figure 2 Fluorescence micrographs of transfected
primary cultures from rat superior colliculus. On day 9 in vitro, cultures
were treated with 200 ng of EGFP plasmid DNA complexed with 5 l of
Effectene Reagent. Cells w
ere incubated with the complexes for 4.5 hours,
the cells were removed, and incubated for a further 24 hours. A
were fixed and stained for MAP2-immunoreactivity which specifically labels
the somatodendritic compartments of neurons (red). B
were stained using DAPI (blue). C
Cells expressing EGFP appear green.
Effectene Transfection Reagent can be used for efficient gene delivery into
differentiated neurons in primary cell culture.
Small amounts of DNA together with Effectene Transfection Reagent delivered
high transfection efficiencies and expression levels, without damaging neurons.
Cell death in 24-well plates was negligible if less than 10 l Effectene
Reagent was used per well and the exposure of cells to transfection complexes
was less than 5 hours.
Effectene Transfection Reagent is a powerful tool for in vitro delivery
of genes to nondividing cells - particularly differentiated neurons - and
therefore greatly facilitates studies of neurophysiology in living cells.
1. Meier, J., Vannier, C.,
Serg, A. Triller, A., and
Choquet, D. (2001) Fast
and reversible trapping of
surface glycine receptors
by gephyrin. Nat.
Neurosci. 4, 253.
2. Meier, J., Meunier-
Durmort, C., Forest, C.,
Triller, A., and Vannier, C.
(2000) Formation of
glycine receptor clusters
and their accumulation at
synapses. J. Cell Sci. 113,
Source:Page: All 1 2 3 4 Related biology technology :1
. Effectene Transfection Reagent2
. Highest Transfection Efficiency of an Endotoxin-Sensitive Mammalian Cell
. Optimizing Transfection Conditions for Studying Signal Transduction Pathways4
. Transfection of Green Fluorescent Protein into Human Adrenalcarcinoma Cells5
. Improve Lipid- or Calcium Phosphate-Mediated Transfection of Human Dermal
. Versatile Transfection Reagent Offers Low Toxicity and Consistent
. Low-Toxicity, Lipid-Mediated Transfection of Mammalian Cells8
. Mycoplasma Contamination Reduces the Effect of Lipid-Mediated Transfection
of Mammalian Cells9
. High-Efficiency Transfections Achieved with New Low-Toxicity Reagent10
. Efficient Transfection of Neurospora Crassa11
. Eppendorf Multiporator Transfection Protocols for Eukaryotic