Protocol

Injection of dsRNA and Electroporation in Avian Embryos

This protocol was adapted from "RNAi in Avian Embryos," contributed by Esther T. Stoeckli, Chapter 14, in RNAi: A Guide to Gene Silencing (ed. Hannon). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2003.

INTRODUCTION

This protocol describes a method for in ovo transfection of avian embryos with double-stranded RNA (dsRNA). The dsRNA is injected into the spinal cord of the embryo. Subsequent electroporation facilitates the cellular uptake of the dsRNA molecules. It may be necessary to optimize the stage of the embryo and the electroporation procedure to improve the effectiveness of in ovo RNAi—cell competence changes with differentiation. The half-life of the target protein product may also affect the time point of injection and electroporation—proteins with slow turnover may require RNAi at earlier stages, preferably before the onset of expression. Check the quality of the dsRNA used for injections and make sure that the buffer used for injection does alter development or survival of the embryo. Tris buffers and buffers containing glycerol are not compatible with in vivo injections. Salt concentration and pH should be in the physiological range. Always use the same buffers for injection and electroporation of test and control embryos.