Use of chick neural tube for optimizing the PSM and epithelial somites electroporation parameters: A detailed protocol.

Muhammad Abu-Elmagd

doi:10.14440/jbm.2018.253

Abstract

Somite myogenesis is one of the crucial early embryonic events that lead to the formation of muscular tissue. A complex of dynamic gene regulatory networks masters this event. To understand and analyze these networks, there remains a genuine need for the use of a reproducible and highly efficient gene transfer technique. In vivo electroporation has proven to be amongst the best approaches in achieving a high level of gene transfer. However, unoptimized electroporation conditions can directly cause varying degrees of cellular damage which may induce abnormal embryonic development as well as changes in the endogenous gene expression. Presegmented mesoderm and epithelial somites are not easy to electroporate. Chick neural tube has served in many functional studies as an ideal experimental model organ which is both robust and easily manipulated. In the current detailed protocol, the neural tube was used as a tool to optimize the electroporation conditions which were subsequently applied in the electroporation of the presegmented mesoderm and epithelial somites. The protocol highlights important notes and hints that enable reproducible results and could be applied in the in vivo electroporation of other chick embryo tissues.

Highlights

Somite formation is a complex of developmentally orchestrated cell signaling that leads to skeletal muscle and vertebral column formation
The current protocol consists of two stages: In the first stage, the neural tube (NT) of HH16 chick embryo (Fig. 6A) was used as a tool to optimize the electroporation conditions
Each voltage was combined with 5 pulses (p), 30 ms pulse space and 100 ms pulse width

Summary

Introduction

Somite formation is a complex of developmentally orchestrated cell signaling that leads to skeletal muscle and vertebral column formation. Gain- and loss-of-gene expression using either in vivo and/or in vitro electroporation is a very powerful approach for manipulating gene function [5] This includes electroporating single (aptamer) [6] or double [7] strand DNA, microRNA inhibitor (antagomir) [8], siRNA [9], morpholino (MO) [10], and Crispr-Cas9 [11] into the whole embryo, the neural tube (NT), neural crest, somites, retinal explants, and brain. Gene transfer by electroporation has few main limitations which include direct damage to the cells, difficulty in controlling the number of electroporated cells, and possible off-target gene transfer. Some of these disadvantages could be controlled through a suitable optimization of the electroporation conditions [12]

Objectives

Methods

Results