A versatile microporation technique for the transfection of cultured CNS neurons.

Published

Journal Article

The application of molecular techniques to cultured central nervous system (CNS) neurons has been limited by a lack of simple and efficient methods to introduce macromolecules into their cytosol. We have developed an electroporation technique that efficiently transfers RNA, DNA and other large membrane-impermeant molecules into adherent hippocampal neurons. Microporation allowed the use of either in vitro transcribed RNA or cDNA to transfect neurons. While RNA transfection yielded a higher percentage of transfected neurons and produced quantitative co-expression of two proteins, DNA transfection yielded higher levels of protein expression. Dextran-based calcium indicators also were efficiently delivered into the cytosol. Microporated neurons appear to survive poration quite well, as indicated by their morphological integrity, electrical excitability, ability to produce action potential-evoked calcium signals, and intact synaptic transmission. Furthermore, green fluorescent protein (GFP)-tagged marker proteins were expressed and correctly localized to the cytosol, plasma membrane, or endoplasmic reticulum. The microporation method is efficient, convenient, and inexpensive: macromolecules can be introduced into most adherent neurons in a 3 mm2 surface area while requiring as little as 1 microl of the material to be introduced. We conclude that the microporation of macromolecules is a versatile approach to investigate signaling, secretion, and other processes in CNS neurons.

Full Text

Cited Authors

  • Teruel, MN; Blanpied, TA; Shen, K; Augustine, GJ; Meyer, T

Published Date

  • October 1999

Published In

Volume / Issue

  • 93 / 1

Start / End Page

  • 37 - 48

PubMed ID

  • 10598863

Pubmed Central ID

  • 10598863

Electronic International Standard Serial Number (EISSN)

  • 1872-678X

International Standard Serial Number (ISSN)

  • 0165-0270

Digital Object Identifier (DOI)

  • 10.1016/s0165-0270(99)00112-0

Language

  • eng