Name: In Ovo Intravascular Injection in Chicken Embryos (Video) | JoVE Journal
Uploaded: 2022-06-03T13:00:00.000+00:00
Duration: 7 min
Description: Learn in ovo intravascular injection techniques for developmental biology research using chicken embryos. This method enables gene transfection, transgenic breeding, and precise delivery of exogenous materials.

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5 minutes

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 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&#39;s body, and the calcium in the hen&#39;s body forms a shell that envelops the fertilized egg, forming a soft-shelled egg in the body. The calcium shell gradually thickens until the egg is produced.
<ol>
	<li>Collect fertilized eggs from a local vendor or institution, which can be stored at 16&#176;C for 1 week. 
	NOTE: The high storage temperature will help the embryo to develop, while the low temperature will decrease the embryo viability. The embryos will fail to develop if the storage time is too long.</li>
	<li>Scrub the eggshell softly and slowly with 75% ethanol before transferring the eggs to the incubator.</li>
	<li>Place the eggs sideways, neatly on a rack in an incubator at 37.8 &#176;C and 60% humidity. After 60 h of incubation (HH stage 17), embryos will develop visible blood vessels and the heart begins to beat15. 
	&#8203;NOTE: After 2 days of incubation, small primordial dots, the early stage of the heart, appear. This process is called cherrybeading. At ~2.5 days, heart and other body segments are formed with visible vessels.</li>
</ol>
2. Preparation before injection
<ol>
	<li>Prepare glass capillaries that will be used as needles. Before the injection, pull 90 mm glass capillaries with an outer diameter of 1 mm and an inner diameter of 0.6 mm using the puller. Break tips using forceps (the angle of glass capillary usually is 45&#176;) and sterilize the capillaries under UV light for 2 h.</li>
	<li>Prepare exogenous materials such as plasmids, packaged lentiviruses, and PGCs.
	<ol>
		<li>Gently mix the plasmid pEGFP-N1 (an enhanced GFP expressing plasmid, 1&#181;g/&#181;L) with liposome at a 1:3 ratio (m/v). Add water to obtain a final DNA concentration of 10 ng/&#181;L. Let the DNA mixture sit at 37 &#176;C for 20 min before injection.</li>
		<li>Thaw viruses on ice before injection. The titer of lentivirus used for injection should be over 5 x 106 Tu/mL.</li>
		<li>Dilute parental or modified gonad PGCs (E4.5, HH stage 24) to 2,000-5,000 cells/&#181;L. 
		&#8203;NOTE: Here we have shown the injection procedure using trypan blue.</li>
	</ol>
	</li>
</ol>
3. Windowing
<ol>
	<li>Before exposing the embryos, sterilize them by gently and softly wiping the surface of the eggshells with 75% alcohol using a cotton ball, ensuring to sterilize the blunt edge of the eggs thoroughly.</li>
	<li>After the sterilization, gently tap the blunt end with forceps to create a small window (0.5 cm x 0.5 cm) on the eggshell surface to expose the embryo. Remove the embryo membrane and then place the egg on the holder under the dissection microscope.</li>
</ol>
4. Injection
<ol>
	<li>Look for blood vessels under the microscope and adjust the focus of the dissection microscope to locate the embryo, and then find the blood vessel ready for injection.</li>
	<li>Fill the glass needle with the exogenous solution (plasmid, lentivirus, or PGCs) using a pipette slowly, avoiding air bubblesin the needle.</li>
	<li>Aim the needle with exogenous solution at the blood vessel, with the needle parallel to the blood vessel being injected.</li>
	<li>Gently insert the filled needle into the vessel and turn on the pump to eject the solution (usually the injected volume is ~1-5 &#181;L) under the microscope. The air pump pressure should be low enough to prevent the destruction of vessels, as the excessively high air pressure would damage blood vessels leading to highspeed flow.
	<ol>
		<li>Once the exogenous solution is injected into the vessel, ensure that the color of the vessel turns into the solution color and recovers to red in 2-5 s, indicating the exogenous solution is successfully delivered to the vessel. At this point the solution enters the artery, and with the beating of the heart is circulated back to the embryo through the artery, and then to the vein. 
		NOTE: There are two injection sites for vascular injection (Figure 1B): one is the head, where the exogenous solution is injected from the vein in the head of the embryo and into the heart through the blood flow, then circulates to the whole embryo with the heart beating (Figure 1B, Arrow 1). Another is through the dorsal aorta of embryo blood; the exogenous solution is injected and spreads to the whole embryo (Figure 1B. Arrow 2) with the beating of the embryonic heart.</li>
	</ol>
	</li>
	<li>After injection, remove the egg from the stereo microscope and drop 200 &#181;L of Penicillin solution inside the eggshell. Cut a 3 cm long piece of medical tape and seal the window. Gently scrape the tape with scissors to squeeze out any air bubbles and then cut another piece to seal the opening across.</li>
	<li>Label and place the injected egg back into the incubator and incubate until the required developmental stage or hatching.</li>
</ol>

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<li>Darnell, D. K., Schoenwolf, G. C. The chick embryo as a model system for analyzing mechanisms of development. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aDarnell%2C+DK+%22Developmental+Biology+Protocols%22">Developmental Biology Protocols</a>. , Humana Press. New York, NY. 25-29 (2000).</li>
<li>Fauzia, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chick+embryo%3A+a+preclinical+model+for+understanding+ischemia-reperfusion+mechanism%5BTitle%5D)+AND+%22Frontiers+in+Pharmacology%22%5BJournal%5D)">Chick embryo: a preclinical model for understanding ischemia-reperfusion mechanism.</a> Frontiers in Pharmacology. 9, 1034(2018).</li>
<li>Fonseca, B. B., da Silva, M. V., de Morais Ribeiro, L. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+chicken+embryo+as+an+in+vivo+experimental+model+for+drug+testing%3A+Advantages+and+limitations%5BTitle%5D)+AND+%22Lab+Animal%22%5BJournal%5D)">The chicken embryo as an in vivo experimental model for drug testing: Advantages and limitations.</a> Lab Animal. 50 (6), 138-139 (2021).</li>
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<li>Islam, M. M., Doh, S. T., Cai, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+ovo+electroporation+in+embryonic+chick+retina%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">In ovo electroporation in embryonic chick retina.</a> Journal of Visualized Experiments. (60), e3792(2012).</li>
<li>Lu, T., Cohen, A. L., Sanchez, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+ovo+electroporation+in+the+chicken+auditory+brainstem%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">In ovo electroporation in the chicken auditory brainstem.</a> Journal of Visualized Experiments. (124), e55628(2017).</li>
<li>van de Lavoir, M. -C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Germline+transmission+of+genetically+modified+primordial+germ+cells%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Germline transmission of genetically modified primordial germ cells.</a> Nature. 441 (7094), 766-769 (2006).</li>
<li>Ballantyne, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Avian+primordial+germ+cells+are+bipotent+for+male+or+female+gametogenesis%5BTitle%5D)+AND+%22Frontiers+in+Cell+and+Developmental+Biology%22%5BJournal%5D)">Avian primordial germ cells are bipotent for male or female gametogenesis.</a> Frontiers in Cell and Developmental Biology. 9, 726827(2021).</li>
<li>Park, T. S., Han, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((piggyBac+transposition+into+primordial+germ+cells+is+an+efficient+tool+for+transgenesis+in+chickens%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences%22%5BJournal%5D)">piggyBac transposition into primordial germ cells is an efficient tool for transgenesis in chickens.</a> Proceedings of the National Academy of Sciences. 109 (24), 9337-9341 (2012).</li>
<li>Lee, H. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeted+gene+insertion+into+Z+chromosome+of+chicken+primordial+germ+cells+for+avian+sexing+model+development%5BTitle%5D)+AND+%22The+FASEB+Journal%22%5BJournal%5D)">Targeted gene insertion into Z chromosome of chicken primordial germ cells for avian sexing model development.</a> The FASEB Journal. 33 (7), 8519-8529 (2019).</li>
<li>Han, J. Y., Lee, B. R. Isolation and characterization of chicken primordial germ cells and their application in transgenesis. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aHan%2C+JY+%22Avian+and+Reptilian+Developmental+Biology%3A+Methods+and+Protocols%22">Avian and Reptilian Developmental Biology: Methods and Protocols</a>. , Humana Press. New York, NY. 229-242 (2017).</li>
<li>Naito, M., Harumi, T., Kuwana, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Long-term+culture+of+chicken+primordial+germ+cells+isolated+from+embryonic+blood+and+production+of+germline+chimaeric+chickens%5BTitle%5D)+AND+%22Animal+Reproduction+Science%22%5BJournal%5D)">Long-term culture of chicken primordial germ cells isolated from embryonic blood and production of germline chimaeric chickens.</a> Animal Reproduction Science. 153, 50-61 (2015).</li>
<li>Yu, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation%2C+characterization+and+germline+chimera+preparation+of+primordial+germ+cells+from+the+Chinese+Meiling+chicken%5BTitle%5D)+AND+%22Poultry+Science%22%5BJournal%5D)">Isolation, characterization and germline chimera preparation of primordial germ cells from the Chinese Meiling chicken.</a> Poultry Science. 98 (2), 566-572 (2019).</li>
<li>Hamburger, V., Hamilton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+series+of+normal+stages+in+the+development+of+the+chick+embryo%5BTitle%5D)+AND+%22Journal+of+Morphology%22%5BJournal%5D)">A series of normal stages in the development of the chick embryo.</a> Journal of Morphology. 88 (1), 49-92 (1951).</li>
<li>Zhang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Crucial+genes+and+pathways+in+chicken+germ+stem+cell+differentiation%5BTitle%5D)+AND+%22The+Journal+of+Biological+Chemistry%22%5BJournal%5D)">Crucial genes and pathways in chicken germ stem cell differentiation.</a> The Journal of Biological Chemistry. 290 (21), 13605-13621 (2015).</li>
<li>Jin, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((UBE2I+stimulates+female+gonadal+differentiation+in+chicken+%28Gallus+gallus%29+embryos%5BTitle%5D)+AND+%22Journal+of+Integrative+Agriculture%22%5BJournal%5D)">UBE2I stimulates female gonadal differentiation in chicken (Gallus gallus) embryos.</a> Journal of Integrative Agriculture. 20 (11), 2986-2994 (2021).</li>
<li>Shi, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((HMGCS1+promotes+male+differentiation+of+chicken+embryos+by+regulating+the+generate+of+cholesterol%5BTitle%5D)+AND+%22All+Life%22%5BJournal%5D)">HMGCS1 promotes male differentiation of chicken embryos by regulating the generate of cholesterol.</a> All Life. 14 (1), 577-587 (2021).</li>
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No conflicts of interest were declared.

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><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&amp;nbsp;</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

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...

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.

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

in-ovo-intravascular-injection-in-chicken-embryos

Research

JoVE Journal

Developmental Biology

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In Ovo Electroporations of HH Stage 10 Chicken Embryos

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In this method, embryonic cardiac tissues are surgically microdissected, dissociated, fluorescently labeled, and implanted into host embryonic tissues. This provides a platform for studying the individual or tissue level developmental organization under ectopic hemodynamic conditions, and/or altered paracrine/juxtacrine environments.

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Learn in ovo intravascular injection techniques for developmental biology research using chicken embryos. This method enables gene transfection, transgenic breeding, and precise delivery of exogenous materials.

In Ovo Intravascular Injection in Chicken Embryos

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