Elsevier

Methods

Volume 39, Issue 3, July 2006, Pages 207-211
Methods

Electroporation of DNA, RNA, and morpholinos into zebrafish embryos

https://doi.org/10.1016/j.ymeth.2005.12.009Get rights and content

Abstract

The combination of accessible embryology and forward genetic techniques has made zebrafish a powerful model system for the study of vertebrate development. One limitation of genetic analysis is that the study of gene function is usually limited to the first developmental event affected by a gene. In vivo electroporation has recently matured as a method for studying gene function at different developmental time points and in specific regions of the organism. The focal application of current allows macromolecules to be efficiently introduced into a targeted region at any time in the life cycle. Here we describe a rapid protocol by which DNA, RNA and morpholinos can all be precisely electroporated into zebrafish in a temporally and spatially controlled manner. This versatile technique allows gene function to be determined by both gain and loss of function analyses in specific regions at specific times. This is the first report that describes the electroporation of three different molecules into embryonic and larval zebrafish cells.

Introduction

Gene transfer techniques have been used extensively in cultured cells, tissues and whole organisms for assaying eukaryotic gene function. Traditionally, the introduction of genes into cells, or into genomes, has relied on transfection, viral infection or microinjection. Ideally, one should be able to study the effects of a gain or loss of gene function effect in the intact, live organism to monitor the effects of genetic modifications in a physiological context. Transgenesis and gene targeting (knock outs) have been invaluable in the assignment of gene function but they present problems when the gene of interest has multiple roles during development. While tissue specific knock outs or inducible expression systems are elegant ways to circumvent this problem, they are expensive, laborious, and limited to certain organisms.

Electroporation has served as an effective method for introducing DNA into bacteria, yeast, and mammalian cells [1]. This technique uses electric pulses to make small holes in the cell membrane through which DNA molecules can enter the cell. The application has been extended in past years to embryos of several species, most notably the chick, facilitating analyses at the molecular level, which traditionally have been difficult to tackle in many model systems [2], [3]. Electroporation into mouse embryos is also possible, though embryos must be cultured after the procedure, a situation that can be extended for a limited time [4]. More recently, Xenopus tadpoles have been electroporated with minimal cell death and excellent survival [5]. Electroporation of embryos has been made feasible by altering voltage and current parameters to minimize damage. Most effective are square wave pulses of low voltage and longer durations compared to conditions used in cultured cells [6], [7], [8]. Electroporation of DNA and RNA into embryos of different stages has thus introduced the possibility of modifying gene activity late in development in organisms that are not genetically tractable. Even when mutations are available, it is often important to analyze the effects of eliminating gene function in a specific tissue while the rest of the animal develops unperturbed. Electroporation provides a means for creating localized loss of function using overexpression of dominant-negative proteins, siRNA [2] or antisense oligonucleotides such as morpholinos [9].

Electroporation of DNA into the zebrafish embryonic neural tube [10] and adult fin [11] have been reported, and this technique was recently used to manipulate Fgf signaling in the zebrafish midbrain/hindbrain region [12]. However, the electroporation of DNA constructs into other tissues, and of mRNA and antisense morpholinos (MOs) into zebrafish, have not been reported. Here we describe a greatly simplified yet precise method for introducing these molecules into neural, retinal, and somitic tissue in the zebrafish embryo. These techniques will extend the use of electroporation and facilitate the analysis of gene function in any tissue and at any time in development.

Section snippets

Embryo preparation and mounting

Zebrafish (Danio rerio) of the Tübingen or TL strains were maintained on a 14–10-h light–dark cycle and bred in our laboratory according to standard conditions [13]. Embryos obtained from mass matings were kept in E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4, and 0.1% methylene blue), staged in hours postfertilization (hpf) [14], dechorionated, and anesthetized using MS222 (Sigma). 20–24 hour embryos were dechorinated by incubating in pronase (125 μg/ml dissolved in E3 medium) for

Concluding remarks

The ability to express or inactivate a gene in a directed fashion is a powerful means of analyzing its role in development. Electroporation is a simple procedure for spatially and temporally regulating gene function that can be performed more easily and quickly than any other transfection method. The electroporation protocol we describe here has several advantages over those previously described for zebrafish. First, by replacing both electrodes with sharpened tungsten needles we were able to

Acknowledgments

We thank Catalina Lafourcade and Judy Bennett for help with fish and members of the Karlstrom laboratory for critically reading the manuscript. This work was supported by a visiting scientist Grant from the CONICYT Bicentennial Program (M.A. and R.O.K.), the Andes Foundation (#C13860), the Fondecyt (1040443 and 1031003) and Millennium Scientific Initiatives (P02-050) (V.P. and M.A.), and NIH NS39994 (R.O.K.).

References (18)

  • H. Nakamura et al.

    Mech. Dev.

    (2004)
  • C.D. Stern

    Dev. Cell

    (2005)
  • T. Saito et al.

    Dev. Biol.

    (2001)
  • K. Haas et al.

    Differentiation

    (2002)
  • T. Suzuki et al.

    FEBS Lett.

    (1998)
  • C. Wolff et al.

    Curr. Biol.

    (2003)
  • B. Sauer

    Methods

    (1998)
  • N. Scheer et al.

    Mech. Dev.

    (1999)
  • F. Andre et al.

    Gene Ther.

    (2004)
There are more references available in the full text version of this article.

Cited by (0)

View full text