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 ...

Hello, I'm Kai Jin from Yangzhou University. On behalf of our research group I'm going to present a powerful tool of the chicken embryo in developmental research. Chicken embryo is a classic developmental model that has been used to investigate development and differentiation, in fact, the laboratory's extraneous measures into chicken embryos is a valuable tool for developmental biology, such as gene transfection, transgenic breeding, and the gonadal transmural preparation.In our lab, we have established the mu-type injection methods but for many researchers, the detail of injection is unclear and hard to line. So in this video, we are going to show the details of vascular injection during early stage of chicken embryo development in ovo. Feel free to contact us if you are interested in it.Here, we show the details of the protocol. Fertilized egg collection and preparation. First, 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, it is incorporated into the funnel of the fallopian tube. As the follicle enters the jugular abdomen of the oviduct, semen binds to the egg in the hen's body and the calcium in the hen's body forms a shell that envelopes the fertilized egg forming a soft shell egg in the body.The calcium shells gradually thicken until the egg is produced. Collect the fertilized egg, academically called embryo eggs and place them neatly on a rack in an incubator at 37.8 degrees Celsius and 60%humidity. The embryos are incubated under suitable conditions.After two day incubations small pulsing red dots called primordial hearts appear. This process is called cherry beating. At around 2.5 days, the blood vessels are visible and the heart and body segments have formed.Needle and plasmid preparation. We use glass capillaries as needles. Before the injection prepare glass capillaries with the outer diameter of one millimeter and the inner diameter of 0.6 millimeters.The exogenous materials usually are plasmids, packaged lentiviruses and primordial germ cells, or PGCs. In this video, we use the trypan blue to show the trace of injected materials.Windowing. Before exposing the embryos, We need to sterilize the embryos to prevent germ contamination.This is done by gently wiping the surface of the embryos with a 75%alcohol cotton ball at the site of the egg opening. After disinfection, gently tap the blunt end of the eggshell with forceps and carefully clip a 0.5 centimeter by 0.5 centimeter hole on the surface of the egg shell with the forceps to expose the embryo. In-ovo intravascular injection.Look at the view under the microscope, adjust the focus of the microscope to first locate the embryo and then find the blood vessels of the chicken embryo ready for injection. Aim the needle with the exogenous substance at the blood vessel, noting that the needle is parallel to the blood vessel being injected. Insert the needle gently into the blood vessel and then turn on the air pump.At this point, the foreign substance enters the artery and with the beating of the heart, circulates back to the embryo through the artery, and then to the vein. There are two types of vascular injections. The one is a head injection where the foreign substance is injected from a vein in the head of the embryo and flows back into the heart.The other is a dorsal aortic injection where the foreign substance circulates to the embryo with the beating of the heart. With the beating of the embryonic heart and the circulation of blood we can clearly see that the foreign material is received by the capillaries at the end of the artery, into the vein and then return to the embryo from the vein, indicating that the foreign material injected is effectively delivered to each part of the embryo. After injection, remove the egg from the stereomicroscope, cut a three centimeter long piece of medical tape with small scissors, and attach the tape to the opening.Gently scrape the tape with the scissors to squeeze out any air bubbles, and then cut another piece of tape to seal the opening into a cross. After sealing the opening, the injected embryos are carefully placed back into the incubator. Here we show the representative images of chicken embryos post injection.The first image shows the result of lentivirus plasmid injection. The green fluorescence under a stereoscopic fluorescence microscope indicates the successful expression of injected plasmid in embryos. This suggests the feasibility of intravascular injection of plasmids into chicken embryos.The second image shows the results of PGC injection. The PGCs were labeled with RFP. In this picture we could see the injected PGCs which are the red points here, located in the recipients'gonad.This indicates that the donor PGCs were able to enter and colonize in the recipients'gonad effectively. In conclusion, this protocol presents the process of in-ovo intravascular injection of exogenous materials, plasmids or PGCs into chicken embryos. The method showed here is easy to follow and learn and worth the application in many fields.This is all I would like to share with you. I hope this approach will facilitate your work as a powerful research tool in setting chicken embryos and the development biology. Thank you for watching this video and good luck with your research.

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5.3K 조회수.  Yangzhou University. 안녕하세요, 저는 양저우 대학의 카이 진입니다. 우리 연구 그룹을 대신하여 나는 발달 연구에서 닭 배아의 강력한 도구를 제시 할 것입니다. 닭 배아는 발달과 분화를 조사하는 데 사용 된 고전적인 발달 모델이며, 실제로 닭 배아에 대한 실험실의 외래 측정은 유전자 형질감염, 형질전환 육종 및 생식선 횡단 준비와 같은 발달 생물학을위한 귀중한 도구입니다.우리 실?...