High-efficiency transfection of mammalian neurons via nucleofection

Zeitelhofer, Manuel; Vessey, John P.; Xie, Yunyun; Tübing, Fabian; Thomas, Sabine; Kiebler, Michael; Dahm, Ralf

doi:10.1038/nprot.2007.226

Cited by 112 publications

(109 citation statements)

References 21 publications

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“…Nucleofection and calcium phosphate co-precipitation have resulted in 75 and 10% transfection in primary rat hippocampal neurons, respectively, but the percent cell death observed after treatment was not stated (68). In differentiated neurons derived from mesencephalic stem cells, however, viability following nucleofection was observed to be 40% (71).…”

Section: Primary Antibodiesmentioning

confidence: 99%

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Functional Protein Delivery into Neurons Using Polymeric Nanoparticles

Hasadsri

Kreuter

Hashimoto³

et al. 2009

Journal of Biological Chemistry

121

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An efficient route for delivering specific proteins and peptides into neurons could greatly accelerate the development of therapies for various diseases, especially those involving intracellular defects such as Parkinson disease. Here we report the novel use of polybutylcyanoacrylate nanoparticles for delivery of intact, functional proteins into neurons and neuronal cell lines. Uptake of these particles is primarily dependent on endocytosis via the low density lipoprotein receptor. The nanoparticles are rapidly turned over and display minimal toxicity to cultured neurons. Delivery of three different functional cargo proteins is demonstrated. When primary neuronal cultures are treated with recombinant Escherichia coli ␤-galactosidase as nanoparticle cargo, persistent enzyme activity is measured beyond the period of nanoparticle degradation. Delivery of the small GTPase rhoG induces neurite outgrowth and differentiation in PC12 cells. Finally, a monoclonal antibody directed against synuclein is capable of interacting with endogenous ␣-synuclein in cultured neurons following delivery via nanoparticles. Polybutylcyanoacrylate nanoparticles are thus useful for intracellular protein delivery in vitro and have potential as carriers of therapeutic proteins for treatment of neuronal disorders in vivo.

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Section: Primary Antibodiesmentioning

confidence: 99%

“…In many cases, uptake efficiency is reported with only a qualitative assessment of toxicity (67)(68)(69). Viral-derived peptides such as the TAT protein transduction domain have only been described as having "highly inefficient" uptake by neurons and "demonstrated toxicity" (70).…”

Section: Primary Antibodiesmentioning

confidence: 99%

Functional Protein Delivery into Neurons Using Polymeric Nanoparticles

Hasadsri

Kreuter

Hashimoto³

et al. 2009

Journal of Biological Chemistry

121

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“…Many currently available transfection methods do not yield sufficiently good gene expression results to allow functional gene analysis in differentiated adherent neurons in vitro. A recent report provides a useful overview of seven different gene delivery methods, including two often-used transfection techniques (calcium-phosphate and lipofection), but excluding magnetofection 6 .…”

Section: Introductionmentioning

confidence: 99%

“…In comparison with the seven recently reviewed methods of gene delivery 6 , we note the following advantages of our magnetofection protocol. First, the method is cost-effective, requires no expensive equipment, is easy to perform, takes only 1 h and does not depend on preparation of complex constructs such as viruses.…”

Section: Introductionmentioning

confidence: 99%

“…Second, it is not toxic for neurons, is used on young as well as mature neurons and allows long-term gene expression. Other methods are preferentially used for young neurons (e.g., 4 DIV 14 ), which are generally much easier to transfect than mature neurons, or, in the case of electroporation and nucleofection 6 , only for cells in suspension at the time of plating. Third, we achieve expression of two mammalian proteins, not only GFP (which is comparatively easy to express), in the same neuron.…”

Section: Introductionmentioning

confidence: 99%

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Efficient transfection of DNA or shRNA vectors into neurons using magnetofection

et al. 2007

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Efficient and long-lasting transfection of primary neurons is an essential tool for addressing many questions in current neuroscience using functional gene analysis. Neurons are sensitive to cytotoxicity and difficult to transfect with most methods. We provide a protocol for transfection of cDNA and RNA interference (short hairpin RNA (shRNA)) vectors, using magnetofection, into rat hippocampal neurons (embryonic day 18/19) cultured for several hours to 21 d in vitro. This protocol even allows double-transfection of DNA into a small subpopulation of hippocampal neurons (GABAergic interneurons), as well as achieving long-lasting expression of DNA and shRNA constructs without interfering with neuronal differentiation. This protocol, which uses inexpensive equipment and reagents, takes 1 h; utilizes mixed hippocampal cultures, a transfection reagent, CombiMag, and a magnetic plate; shows low toxicity and is suited for single-cell analysis. Modifications done by our three laboratories are detailed. INTRODUCTIONEvery gene transfer method aims to achieve high transfection efficiency, low toxicity and long-lasting expression. These goals are especially difficult to achieve for postmitotic primary neurons due to their high sensitivity to any microenvironmental change 1-5 . Many currently available transfection methods do not yield sufficiently good gene expression results to allow functional gene analysis in differentiated adherent neurons in vitro. A recent report provides a useful overview of seven different gene delivery methods, including two often-used transfection techniques (calcium-phosphate and lipofection), but excluding magnetofection 6 . Magnetofection is a technique that can be used to reliably and efficiently introduce DNA into a variety of cell types 7 . Therefore, we have invested in designing a reliable, magnetofection-based protocol to express DNA or shRNA constructs in rat hippocampal neurons (embryonic day (E)18/19) cultured for several hours to 21 d in vitro (DIV). After transfection, neurons expressing exogenous proteins can develop normally in culture for 5-10 d. We have determined the optimum parameters for transfection efficiency such as age of cells at transfection time point, expression levels during the time after transfection and parameters for doubletransfection. This protocol shows low toxicity, uses a combination of a transfection reagent, CombiMag, and magnetic force and shows a sufficiently high efficiency in neurons (ca. 5%) to allow single-cell analysis.We present a core protocol and illustrate the critical steps, modifications and applications carried out by our three laboratories (Medina, Fuhrer and Fritschy) [8][9][10] . This protocol allows single and double transfections of DNA vectors at different time points after plating. First, we present a detailed characterization of magnetofection parameters such as age of neurons, time course of protein expression after magnetofection, and transfection efficiency in pyramidal neurons and GABAergic interneurons. Three specific

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Transfection of Cultured Primary Neurons via Nucleofection

et al. 2009

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Despite the development of various transfection methods, the transfection of post-mitotic cells, including neurons, poses a challenging task. Nucleofection, a specialized form of electroporation described in this unit, achieves high transfection efficiencies in primary mammalian neurons, such as hippocampal neurons, while simultaneously maintaining high cell viability. Therefore, it allows for biochemical analyses that rely on large numbers of transfected cells. The recently developed 96-well shuttle system described in this unit further permits the transfection of up to 96 different constructs in a single experiment. This opens up the possibility for large-scale experiments in primary neurons, such as shRNA-mediated knock-down of a wide range of target genes.

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High-efficiency transfection of mammalian neurons via nucleofection

Cited by 112 publications

References 21 publications

Functional Protein Delivery into Neurons Using Polymeric Nanoparticles

Functional Protein Delivery into Neurons Using Polymeric Nanoparticles

Efficient transfection of DNA or shRNA vectors into neurons using magnetofection

Transfection of Cultured Primary Neurons via Nucleofection

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