In Ovo Intravascular Injection in Chicken Embryos

Summary

The overall goal of this paper is to describe how to perform in ovo intracellular injection of exogenous materials into chicken embryos. This approach is very useful to study the developmental biology of chicken embryos.

Abstract

As a classical model system of embryo biology, the chicken embryo has been used to investigate embryonic development and differentiation. Delivering exogenous materials into chicken embryos has a great advantage for studying gene function, transgenic breeding, and chimera preparation during embryonic development. Here we show the method of in ovo intravascular injection whereby exogenous materials such as plasmid vectors or modified primordial germ cells (PGCs) can be transferred into donor chicken embryos at early developmental stages. The results show that the intravascular injection through the dorsal aorta and head allows injected materials to diffuse into the whole embryo through the blood circulatory system. In the presented protocol, the efficacy of exogenous plasmid and lentiviral vector introduction, and the colonization of injected exogenous PGCs in the recipient gonad, were determined by observing fluorescence in the embryos. This article describes detailed procedures of this method, thereby providing an excellent approach to studying gene function, embryo and developmental biology, and gonad-chimeric chicken production. In conclusion, this article will allow researchers to perform in ovo intravascular injection of exogenous materials into chicken embryos with great success and reproducibility.

Introduction

Chicken embryos have been widely used for centuries in developmental, immunological, pathological, and other biological applications^1,2,3. They have many inherent advantages over other animal models in the study of toxicology and cell biology⁴. Chicken embryos are easily accessible and can be manipulated in vitro and directly observed at any developmental stage, which provides a handy embryo research model system.

In general, current chicken embryo delivery methods such as electrotransfection and subgerminal-cavity ....

Protocol

All procedures involving the care and use of animals conformed to U.S. National Institute of Health guidelines (NIH Pub. No. 85-23, revised 1996) and the chicken embryo protocols were approved by the Laboratory Animal Management and Experimental Animal Ethics Committee of Yangzhou University, China (No.201803124).

1. Fertilized egg collection and preparation

NOTE: Unlike mammals, the chicken has millions of follicles in a single ovary, but only a few of these follicles are mature enough to ovulate. Each follicle contains one oocyte or germ cell. As soon as the follicle matures and releases its yolk....

Results

We show here the in ovo intravascular injection of chicken embryos. A schematic process of the intravascular injection is shown in Figure 1; in our study, we used various exogenous solutions to test and verify injection.

To better visualize the injected materials, Trypan Blue (0.4%) was injected as a tracer into the embryo. The tracer (blue) was observed to diffuse to the whole embryo via blood circulation by either dorsal aorta or head injection.......

Discussion

The method of in ovo intravascular injection of chicken embryos is optimized for exogenous materials (vector, viral, or PGCs) to be transferred into the embryo. Based on this method, we constructed chicken embryo models with stable gene overexpression or interference (SpinZ, JUN, UBE2I, etc.)^17,18,19. These well-established models prove the feasibility of this approach. Additionally, we not only transferred isolated PGC.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Fluorescence macro-microscope	OLYMPUS	MVX10
Glass Capillaries	Narishige	G1
Lipofectamine 2000	Invitrogen	12566014	liposome
pEGFP-N1 vector	Clontech	#6085-1
PKH26 Red Fluorescent Cell Linker Kit	Sigma	PKH26GL
pLVX-EGFP lentivirus vector	Addgene	128652
Pneumatic Microinjector	Narishige	IM-11-2
Puller	Narishige	PC-100
Trypan Blue Stain	Gibco	15250061