A Simple, Highly Efficient Method for Heterologous Expression in Mammalian Primary Neurons Using Cationic Lipid-mediated mRNA Transfection

Damian J Williams,Henry L Puhl,Stephen R Ikeda

doi:10.3389/fnins.2010.00181

Abstract

Expression of heterologous proteins in adult mammalian neurons is a valuable technique for the study of neuronal function. The post-mitotic nature of mature neurons prevents effective DNA transfection using simple, cationic lipid-based methods. Adequate heterologous protein expression is often only achievable using complex techniques that, in many cases, are associated with substantial toxicity. Here, a simple method for high efficiency transfection of mammalian primary neurons using in vitro transcribed mRNA and the cationic lipid transfection reagent Lipofectamine™ 2000 is described. Optimal transfection conditions were established in adult mouse dissociated dorsal root ganglion (DRG) neurons using a 96-well based luciferase activity assay. Using these conditions, a transfection efficiency of 25% was achieved in DRG neurons transfected with EGFP mRNA. High transfection efficiencies were also obtained in dissociated rat superior cervical ganglion (SCG) neurons and mouse cortical and hippocampal cultures. Endogenous Ca2+ currents in EGFP mRNA-transfected SCG neurons were not significantly different from untransfected neurons, which suggested that this technique is well suited for heterologous expression in patch clamp recording experiments. Functional expression of a cannabinoid receptor (CB1R), a G protein inwardly rectifying K+ channel (GIRK4) and a dominant-negative G protein α-subunit mutant (GoA G203T) indicate that the levels of heterologous protein expression attainable using mRNA transfection are suitable for most functional protein studies. This study demonstrates that mRNA transfection is a straightforward and effective method for heterologous expression in neurons and is likely to have many applications in neuroscience research.

Highlights

Heterologous protein expression in primary neurons is a valuable technique for the study of neuronal function, which allows the roles of specific proteins to be studied in a physiologically relevant environment
As described in the introduction, cationic lipid transfection of mRNA has potential advantages for heterologous expression in non-dividing cells. For this reason a series of experiments were undertaken to establish the optimal conditions for mRNA transfection of dissociated dorsal root ganglion (DRG) neurons
Neurons were transfected with Rluc mRNA and luciferase expression was quantified from cell lysates using a luminescencebased luciferase activity assay

Summary

Introduction

Heterologous protein expression in primary neurons is a valuable technique for the study of neuronal function, which allows the roles of specific proteins to be studied in a physiologically relevant environment. Heterologous expression from plasmid cDNA vectors using simple cationic lipid-based transfection methods are effective in dividing cells, but often result in very low transfection efficiencies in post-mitotic cells such as mature neurons. The absence of a simple and effective method for heterologous protein expression in neurons restricts many experiments to other models such as cell lines, which may lack many of the characteristics of primary neurons. Development of such a method would be a valuable tool for neuroscience research

Methods

Results

Conclusion