Sonoporation for Gene Transfer into Embryos

INTRODUCTION

Sonoporation is a method for the introduction of genes into tissues such as those of chick embryos. It enables gene incorporation into target tissue by the combination of ultrasound exposure and subsequent rupture of microbubbles. Sonoporation has several advantages: (1) It is simple, involving two main steps—preparation of a microbubble–DNA mixture, followed by injection and ultrasound treatment of the target tissue; (2) it allows highly efficient gene incorporation into mesenchymal tissues; (3) it does not induce significant tissue damage—most of the sonoporated chick embryos survived without showing significant embryonic abnormalities or cell death; and (4) it can be used to introduce genes into several chick embryonic tissues (e.g., branchial arch and lateral plate mesoderm). To demonstrate the utility of sonoporation, an expression vector containing the Sonic hedgehog (Shh) gene was incorporated into the developing chick limb bud, leading to additional digit formation in the Shh-expressing embryos. Sonoporation may not be appropriate for the introduction of genes into tissues with cavitated or open structures. Sonoporation into the neural tube and limb ectoderm often resulted in dispersed gene expression due to diffusion of the injected microbubbles before ultrasound treatment. Despite these problems, sonoporation is a useful method for the analysis of gene function(s) in vivo and brings a new area of gene transfer to medical, molecular, and developmental biology. This protocol describes the use of sonoporation to introduce vectors into embryonic chick limb bud.