Biolistic transfection and expression analysis of acute cortical slices

Mohammad I.K. Hamad, Solieman Daoud, Petya Petrova, Obada Rabaya, Abdalrahim Jbara, Nesrine Melliti, Sarah Stichmann, Gebhard Reiss, Joachim Herz, Eckart Förster

Research output: Contribution to journalArticle

Abstract

Background: Biolistic gene gun transfection has been used to transfect organotypic cultures (OTCs) or dissociated cultures in vitro. Here, we modified this technique to allow successful transfection of acute brain slices, followed by measurement of neuronal activity within a few hours. New method: We established biolistic transfection of murine acute cortical slices to measure calcium signals. Acute slices are mounted on plasma/thrombin coagulate and transfected with a calcium sensor. Imaging can be performed within 4 h post transfection without affecting cell viability. Results: Four hours after GCaMP6s transfection, acute slices display remarkable fluorescent protein expression level allowing to study spontaneous activity and receptor pharmacology. While optimal gas pressure (150 psi) and gold particle size used (1 μm) confirm previously published protocols, the amount of 5 μg DNA was found to be optimal for particle coating. Comparison with existing methods: The major advantage of this technique is the rapid disposition of acute slices for calcium imaging. No transgenic GECI expressing animals or OTC for long periods are required. In acute slices, network interaction and connectivity are preserved. The method allows to obtain physiological readouts within 4 h, before functional tissue modifications might come into effect. Limitations of this technique are random transfection, low expression efficiency when using specific promotors, and preclusion or genetic manipulations that require a prolonged time before physiological changes become measurable, such as expression of recombinant proteins that require transport to distant subcellular localizations. Conclusion: The method is optimal for short-time investigation of calcium signals in acute slices.

Original languageEnglish (US)
Article number108666
JournalJournal of Neuroscience Methods
Volume337
DOIs
StatePublished - May 1 2020

Keywords

  • Acute slices
  • Calcium imaging
  • Gene-gun transfection
  • Genetically encoded calcium indicators
  • Neocortex

ASJC Scopus subject areas

  • Neuroscience(all)

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  • Cite this

    Hamad, M. I. K., Daoud, S., Petrova, P., Rabaya, O., Jbara, A., Melliti, N., Stichmann, S., Reiss, G., Herz, J., & Förster, E. (2020). Biolistic transfection and expression analysis of acute cortical slices. Journal of Neuroscience Methods, 337, [108666]. https://doi.org/10.1016/j.jneumeth.2020.108666