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Introducing Cloned Genes into Cultured Neurons Providing Novel In vitro Models for Neuropathology and Neurotoxicity Studies

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Cell Culture Techniques

Part of the book series: Neuromethods ((NM,volume 56))

Abstract

Recent advances in techniques to introduce nucleic acids into cultured cells have significantly contributed to understanding the roles of genes (and their encoded proteins) in maintaining cellular homeostasis. The objective of this chapter is to provide methodological strategies for gene introduction specifically into cultured neuronal cells. This approach has been used to study the role of specific proteins in neurodegenerative and neuroprotective events, as well as in neurotransmission, antioxidant defenses, energetic metabolism, and several other physiological phenomena related to the neuronal homeostasis. The chapter starts with a description of the most important vectors currently available for neuronal transfections. A particular emphasis is directed at plasmid vectors, and a simple but useful protocol to isolate plasmids from bacteria is presented. This is followed by a discussion on the fundamentals of gene manipulation emphasizing the basics on how to isolate a DNA fragment, as well as modify and insert it into a vector. Since bacteria can be transfected with the cloning vector, it is possible to achieve high levels of the vector during bacterial growth. The purified vector can be inserted into a eukaryotic cell, such as a neuron, which uses its transcriptional machinery to overexpress the protein of interest. The chapter also presents discussions and protocols on delivering nucleic acids into cultured neuronal cells (primary and cell lines), with a particular emphasis on lipid-based (lipofection) and electroporation-based transfection. At the end of the chapter, we discuss recent applications of gene transfection to study neuropathology and neurotoxicity. The use of strategies to overexpress specific proteins into cultured neuronal cells has been useful to study neurodegenerative diseases (i.e., Parkinson disease vs. alpha-synuclein or parkin) and neurotoxicity events (i.e., methylmercury-induced neurotoxicity vs. glutathione peroxidase). In this regard, studies point to the fact that genetically-modified cultured neuronal cells may help neurotoxicologists in the difficult task of screening environmental toxicants with potential hazard for predisposition to neurodegenerative diseases.

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Acknowledgments

The authors would like to thank the agencies that support their research activities (in alphabetical order): Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq and INCT for Excitotoxicity and Neuroprotection-MCT/CNPq, Brazil (Marcelo Farina and João B. T. Rocha); Fundação de Amparo à Pesquisa do Estado de Santa Catarina, Brazil (Marcelo Farina); NIH ES10563 and ES 07331 (Michael Aschner); Spanish Ministry of Education Grant BFU2008-02424/BFI (Sebastián Pons). Jordi Berenguer is a FPI fellow in Sebastián Pons laboratory (fellowship also supported by the Spanish Ministry of Education Grant BFU2008-02424/BFI).

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Authors and Affiliations

  1. Departamento de Bioquímica, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Florianópolis, Santa Catarina, Brazil

    Marcelo Farina

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  1. Marcelo Farina
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  2. Jordi Berenguer
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  3. Sebastián Pons
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  4. João Batista Teixeira da Rocha
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  5. Michael Aschner
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Corresponding author

Correspondence to Marcelo Farina .

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Editors and Affiliations

  1. , Department of Pediatrics, Vanderbilt University Medical Center, Garland Avenue 2215-B, Nashville, 37232, Tennessee, USA

    Michael Aschner

  2. Institut d’Investigacions Biomèdiques, Neurochemistry & Neuropharmacology, CSIC, C/ Rosselló 161, Barcelona, 08036, Spain

    Cristina Suñol

  3. Inst. for Health & Consumer Protection, ECVAM In-Vitro Methods Unit, European Commission JRC, Via Enrico Fermi 1, Ispra, 21026, Varese, Italy

    Anna Bal-Price

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Farina, M., Berenguer, J., Pons, S., da Rocha, J.B.T., Aschner, M. (2011). Introducing Cloned Genes into Cultured Neurons Providing Novel In vitro Models for Neuropathology and Neurotoxicity Studies. In: Aschner, M., Suñol, C., Bal-Price, A. (eds) Cell Culture Techniques. Neuromethods, vol 56. Humana Press. https://doi.org/10.1007/978-1-61779-077-5_9

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  • DOI: https://doi.org/10.1007/978-1-61779-077-5_9

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-076-8

  • Online ISBN: 978-1-61779-077-5

  • eBook Packages: Springer Protocols

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