Review - amaxa eNews #4

amaxa eNews #4

High-efficiency Transfection Offers New Possibilities in Neuroscience Research

Introduction

Exploring the function of the many thousands of mRNAs within neural transcriptomes is a major challenge in current neuroscience research. Molecular methods applied promise to significantly contribute to overcome this challenge and gain a better scientific understanding of neural genes.

For many cell types, the introduction of nucleic acids and other substrates has become a ground-breaking tool to explore gene function. For example, changes in the molecular equipment of a cell induced by the introduction of different molecules not only enable the dissection of signal transduction [1] and metabolic pathways, but also the analysis of local RNA translation [2]. Furthermore, the transfection of fluorescently labelled or tagged proteins has been used to explore the activities of proteins inside living cells or for isolation of proteins by immunoprecipitation or pull-down assays, respectively. With the advent of RNA interference (RNAi), the transfection of siRNAs [3] or shRNA constructs [4] has become one of the most exciting methods for gene identification and functional validation in eukaryotic cells [5].

However, although a huge variety of transfection methods exists for immortalized cell lines, traditional non-viral transfection methods are often ineffective, toxic, or both when applied to post-mitotic cells such as primary neurons. Low transfection efficiencies or no transfection is common using technolgies such as lipofection with primary neurons. Viral-mediated gene transfer, although highly efficient, is often quite time-consuming and has safety issues. An overview of the benefits and drawbacks of the different transfection techniques is given in Table 1 [6].

Relevance of high-efficiency transfection in neuroscience

Low transfection efficiencies may be acceptable in some cases as stable clone generation may be the goal, although this is usually a time consuming and tedious procedure, and is not an option for primary neurons.

With primary neurons, low transfection efficiencies can be a critical experimental obstacle, making many downstream applications impossible [7]. For instance, in RNAi experiments highly efficient delivery of siRNA molecules or shRNA encoding plasmids is an important prerequisite in obtaining functionally relevant and detectable gene silencing.

For many neuron types such as DRGs, retinal, or cerebellar granule neurons, transfection efficiencies obtained by conventional methods are very low and satisfactory gene silencing often cannot be detected for single cells by microscopy (e.g. by analysis of cellular morphology). If biochemical assays for the analysis of gene silencing are needed, such as western blot for the analysis of silencing at the protein level, it is crucial that a large proportion of cells is transfected in order to have a significant signal in comparison to background. High numbers of cells with little or no gene silencing lead to high background signals for the target protein. This is problematic since the resolution of biochemical methods may not be sufficient to detect gene silencing although gene silencing might occur in individual cells [6]. High transfection efficiencies are a crucial prerequisite for successful biomolecular experiments in neuroscience research.

An additional challenge in neuroscience research is that the number of neurons that can be obtained from the preparation of a donor animal is extremely low. For instance, just 100,000 to 300,000 DRG cells are typically obtained from one donor rat. Excellent transfection efficiencies and reproducibilities are crucial in order to generate as much experimental data as possible from the given number of these very precious cells. Besides it is useful to be able to transfect just the number of cells required for the intended downstream analysis. For microscopic analyses for instance, the transfection of neurons on a single cover slip, i.e. typically 15,000-100,000 cells, can be desired.

New possibilities enabled by Nucleofector Technology

Nucleofector Technology overcomes the limitations of conventional transfection techniques because it enables direct delivery of DNA, siRNA oligonucleotides and RNAi vectors into the cytoplasm and nucleus of non-dividing cells such as primary neurons. This strategy results in reproducible, rapid, and efficient transfection of a broad range of neural cells. Nucleofection requires considerably fewer steps than other transfection protocols and completely lacks the toxic side effects associated with chemical transfection reagents. Because of its simplicity, nucleofection tends to give highly reproducible results, which makes nucleofection attractive as a primary experimental tool.

The new small-cell-number (SCN) Nucleofector Kit makes high-efficiency transfection possible even when using down to 15,000 neurons per reaction. This allows you to do more experiments using fewer neuronal cells, which are typically present in low abundance. Using the SCN Nucleofector Kit it is possible to transfect only the cells you need for a single cover slip.

Finally, for applications demanding high throughput transfection of neural cells, amaxa’s 96-well Shuttle represents a solid tool for rapid and inexpensive screening approaches, such as RNAi library screening.

Around 200 publications prove the Nucleofector Technology to be an excellent research tool for a broad range of neural cell types including primary neurons, glial cells such as astrocytes or oligodendroytes, and neural cell lines.

Transfection Method	Advantages	Disadvantages
Viral methods	High transfection efficiency	Generation of viral particles is time consuming
	Low neuron mortality	Health risk due to potential infectiousness of viruses
		Extensive safety requirements

Lipofection	Easy handling	Induces very high neuron mortality
	Applicable to adherent neuron cultures (though very inefficient)	Neurons frequently suffer from drastic morphological changes

Ca²⁺-phosphate	Cheap reagent	Very low transfection efficiencies
	Low neuron mortality	Very low reproducibility

Electroporation	High transfection efficiencies	Freshly isolated neurons only
		High mortality
		Electroporator required

Microinjection	Injection of directly labelled RNA or oligonucleotides possible	Low transfection efficiency, (very much dependent on neuron type and researcher´s experience)
		Extremely high mortality
		Expensive equipment

Biolistics	Applicable for tissue slices	High mortality
		Neurons frequently suffer from drastic morphological changes
		Specialized equipment required

Nucleofection	Non-viral, i.e. no virus-related health risk or safety requirements	Specialized equipment required
	High transfection efficiency	Freshly isolated neurons only
	Low neuron mortality
	Long term survival without loss of functionality
	Ready-to-use kits and protocols with detailed culture information for most common neural cell types

Conclusion

The ability to transfect neural cells with high efficiency, reproducibility and even at low cell numbers is an important prerequisite for adapting current biomolecular applications to primary neurons.

By providing this ability, amaxa’s Nucleofector Technology offers new research opportunities, for example, in the area of gene silencing. Due to limitations of common methods, RNAi experiments have been mostly performed in easy-to-transfect cell lines. Thus, primary cells such as neurons have been somewhat neglected. With the Nucleofector Technology, successful RNAi experiments are now possible in primary neurons.

In summary, future experiments enabled by highly efficient delivery of nucleic acids to neural cells using the Nucleofector Technology promise to significantly increase our understanding of the cellular and molecular mechanisms in neural development, physiology and function.

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References

[1] Heim et al. (2007) Improved calcium imaging in transgenic mice expressing a troponin C-based biosensor. Nat. Methods 4, 127–129

[2] Hüttelmaier et al. (2005) Spatial regulation of beta-actin translation by Src-dependent phosphorylation of ZBP1. Nature 438, 512–515.

[3] Schratt et al. (2006) A brain-specific microRNA regulates dendritic spine development. Nature 439, 283–289.

[4] Zeringue et al. (2004) Post-transcriptional gene silencing in neurons. Curr. Opin. Neurobiol. 14, 654–659.

[5] Müller-Hartmann et al. (2007) High-throughput transfection and engineering of primary cells and cultured cell lines – an invaluable tool for research as well as drug development. Expert Opin. Drug Discov. 2(11)

[6] Zeitelhofer et al. (2007) High-efficiency transfection of mammalian neurons via nucleofection. Nat Protoc. 2(7):1692-704.

[7] Dahm et al (2008): Visualising mRNA localisation and local protein translation in neurons. Methods in Cell Biology (in press).

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Mouse DRG neuron nucleo- fected with pmaxGFP, 24h post nucleofection.
Data courtesy of B. Eickholt, MRC Centre for Developmental Neurobiology, King‘s College, London, United Kingdom.

The Nucleofector Technology, comprising Nucleofection Process, Nucleofector Device, Nucleofector Solutions, Nucleofector 96-well Shuttle System and Nucleocuvette plates and modules is covered by patent and/or patent pending rights owned by amaxa AG.

amaxa, Nucleofector, nucleofection, maxGFP, 96-well Shuttle and Nucleocuvette are either registered trademarks or trademarks of amaxa AG in the U.S. and/or Germany and/or other countries. Other product and company names mentioned herein are the trademarks of their respective owners.

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