Name: in ovo intravascular injection in chicken embryos
Uploaded: 2022-06-03T13:00:00
Description: Watch this Scientific Journal Video about In Ovo Intravascular Injection in Chicken Embryos at JoVE.com

This work was supported by the National Natural Science Foundation of China (31972547). We appreciate the copyediting by Jing Wang and the voiceover by Malik Donlic at Washington State University, USA.

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 ...

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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...

Chicken embryos have been widely used for centuries in developmental, immunological, pathological, and other biological applications1,2,3. 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 injection have limitations such as the requirement of specialist equipment and a designed program, and inefficiency due to the presence of yolk and albumen5,6,7. Here we show a simple and efficient handling method for delivering exogenous materials into chicken embryos. This can be a powerful tool used in the study of developmental biology. The injected materials spread to the whole embryo via blood circulation. During the early development of chicken embryos, the PGCs could migrate through blood, colonize the genital ridge, and then develop into gametes, which provide a valuable possible path to deliver exogenous materials8. Now, this method has been widely used in the study of gene function, embryo and developmental biology, and chimeric and transgenic chicken production9,10,11.
In ovo intravascular injection in chicken embryos is a well-established and commonly used method12,13,14. In this paper, we show a comprehensive description of this protocol including injection materials, sites, dosage, and representative results.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Fluorescence macro-microscope</td>
			<td>OLYMPUS</td>
			<td>MVX10</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Capillaries</td>
			<td>Narishige</td>
			<td>G1</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Invitrogen</td>
			<td>12566014</td>
			<td>liposome</td>
		</tr>
		<tr>
			<td>pEGFP-N1 vector</td>
			<td>Clontech</td>
			<td>#6085-1</td>
			<td></td>
		</tr>
		<tr>
			<td>PKH26 Red Fluorescent Cell Linker Kit&#160;</td>
			<td>Sigma</td>
			<td>PKH26GL</td>
			<td></td>
		</tr>
		<tr>
			<td>pLVX-EGFP lentivirus vector</td>
			<td>Addgene</td>
			<td>128652</td>
			<td></td>
		</tr>
		<tr>
			<td>Pneumatic Microinjector</td>
			<td>Narishige</td>
			<td>IM-11-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Puller</td>
			<td>Narishige</td>
			<td>PC-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Stain</td>
			<td>Gibco</td>
			<td>15250061</td>
			<td></td>
		</tr>
	</tbody>
</table>

in ovo intravascular injection in chicken embryos

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.

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...

Watch this Scientific Journal Video about In Ovo Intravascular Injection in Chicken Embryos at JoVE.com

In Ovo Intravascular Injection in Chicken Embryos

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.

In Ovo Intravascular Injection in Chicken Embryos

As a classical model system of embryo biology, the chicken embryo has been used to investigate embryonic development and differentiation. Delivering ...

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