Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.

Summary

Microinjection is a well-established and effective method for introducing foreign substances into fertilized zebrafish embryos. Here, we demonstrate a robust microinjection technique for performing mRNA overexpression, and morpholino oligonucleotide gene knockdown studies in zebrafish.

Abstract

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression studies. In zebrafish (Danio rerio), microinjection of RNA, DNA, proteins, antisense oligonucleotides and other small molecules into the developing embryo provides researchers a quick and robust assay for exploring gene function in vivo. In this video-article, we will demonstrate how to prepare and microinject in vitro synthesized EGFP mRNA and a translational-blocking morpholino oligo against pkd2, a gene associated with autosomal dominant polycystic kidney disease (ADPKD), into 1-cell stage zebrafish embryos. We will then analyze the success of the mRNA and morpholino microinjections by verifying GFP expression and phenotype analysis. Broad applications of this technique include generating transgenic animals and germ-line chimeras, cell-fate mapping and gene screening. Herein we describe a protocol for overexpression of EGFP and knockdown of pkd2 by mRNA and morpholino oligonucleotide injection.

Protocol

Part 1: Preparation of micropipettes, and microinjection chamber plates

1. Fabricate micropipettes by heating and pulling borosilicate glass capillary tubes (World Precision Instruments, Inc., 1B100-4) in a micropipette puller device (Sutter Instruments Inc., Flaming/Brown P-97). Store in a Petri dish on top of small amount of clay or adhesive tape.
2. Pour 1.5% agarose (American Bioanlytical, Inc., AB00972-00500) in 1x E3 medium plates molded with wedge-shaped troughs that will serve as an easy method for holding the embryos during the injection (as described in 'The Zebrafish Book") ¹. Let agar chamber plates solidify before use a....

Discussion

Microinjection into zebrafish embryos is a well-established and robust technique for exploring the role of a particular gene in development. Applications include overexpression, misexpression, and knockdown assays of your gene of interest as well as epistatic analysis between multiple genes. Microinjection in zebrafish has been widely used for generating transgenic animals, and mapping cell fate in early blastula embryos^5,6,7,8,9. In addition, the application of this technique serves as a key step in generatin.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1x E3 Medium	Reagent			5 mM NaCl 0.17 mM KCl 0.33 mM CaCl2 0.33 mM MgSO2 0.1% Methylene blue
All other materials are listed in the protocol.