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>

<ol>
	<li>Bednarczyk, M., Dunislawska, A., Stadnicka, K., Grochowska, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chicken+embryo+as+a+model+in+epigenetic+research.">Chicken embryo as a model in epigenetic research.</a> Poultry Science. 100 (7), 101164 (2021).</li><li>Darnell, D. K., Schoenwolf, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chick+embryo+as+a+model+system+for+analyzing+mechanisms+of+development.">The chick embryo as a model system for analyzing mechanisms of development.</a> Developmental Biology Protocols. , 25-29 (2000).</li><li>Fauzia, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chick+embryo:+a+preclinical+model+for+understanding+ischemia-reperfusion+mechanism.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chicken+embryo+as+an+in+vivo+experimental+model+for+drug+testing:+Advantages+and+limitations.">The chicken embryo as an in vivo experimental model for drug testing: Advantages and limitations.</a> Lab Animal. 50 (6), 138-139 (2021).</li><li>Blank, M. C., Chizhikov, V., Millen, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+ovo+electroporations+of+HH+stage+10+chicken+embryos.">In ovo electroporations of HH stage 10 chicken embryos.</a> Journal of Visualized Experiments. (9), e408 (2007).</li><li>Islam, M. M., Doh, S. T., Cai, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+ovo+electroporation+in+embryonic+chick+retina.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+ovo+electroporation+in+the+chicken+auditory+brainstem.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germline+transmission+of+genetically+modified+primordial+germ+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Avian+primordial+germ+cells+are+bipotent+for+male+or+female+gametogenesis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=piggyBac+transposition+into+primordial+germ+cells+is+an+efficient+tool+for+transgenesis+in+chickens.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+insertion+into+Z+chromosome+of+chicken+primordial+germ+cells+for+avian+sexing+model+development.">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. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+chicken+primordial+germ+cells+and+their+application+in+transgenesis.">Isolation and characterization of chicken primordial germ cells and their application in transgenesis.</a> Avian and Reptilian Developmental Biology: Methods and Protocols. , 229-242 (2017).</li><li>Naito, M., Harumi, T., Kuwana, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+culture+of+chicken+primordial+germ+cells+isolated+from+embryonic+blood+and+production+of+germline+chimaeric+chickens.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+characterization+and+germline+chimera+preparation+of+primordial+germ+cells+from+the+Chinese+Meiling+chicken.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+series+of+normal+stages+in+the+development+of+the+chick+embryo.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crucial+genes+and+pathways+in+chicken+germ+stem+cell+differentiation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UBE2I+stimulates+female+gonadal+differentiation+in+chicken+(Gallus+gallus)+embryos.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HMGCS1+promotes+male+differentiation+of+chicken+embryos+by+regulating+the+generate+of+cholesterol.">HMGCS1 promotes male differentiation of chicken embryos by regulating the generate of cholesterol.</a> All Life. 14 (1), 577-587 (2021).</li><li>Jiang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spin1z+induces+the+male+pathway+in+the+chicken+by+down-regulating+Tcf4.">Spin1z induces the male pathway in the chicken by down-regulating Tcf4.</a> Gene. 780, 145521 (2021).</li><li>Zhao, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+viable+chicken+by+allogeneic+transplantation+of+primordial+germ+cells+induced+from+somatic+cells.">Production of viable chicken by allogeneic transplantation of primordial germ cells induced from somatic cells.</a> Nature Communications. 12 (1), 2989 (2021).</li></ol>

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

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 ...

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

Research

JoVE Journal

Developmental Biology

5.6K Views.  Yangzhou University. 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.

Video: In Ovo Intravascular Injection in Chicken Embryos

In Ovo Electroporations of HH Stage 10 Chicken Embryos

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology.  With the advent of molecular biological techniques, the chick has become a useful system in which to study gene regulation and function.  By electroporating DNA or RNA constructs into the developing chicken embryo, genes can be expressed or knocked down in order to analyze in vivo gene function.  Similarly, reporter constructs can be used for fate mapping or to examine putative gene regulatory elements.  Compared to similar experiments in mouse, chick electroporation has the advantages of being quick, easy and inexpensive.  This video demonstrates first how to make a window in the eggshell to manipulate the embryo.  Next, the embryo is visualized with a dilute solution of India ink injected below the embryo.  A glass needle and pipette are used to inject DNA and Fast Green dye into the developing neural tube, then platinum electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator.  Finally, the egg is sealed with tape and placed back into an incubator for further development.  Additionally, the video shows proper egg storage and handling and discusses possible causes of embryo loss following electroporation.

Chick in ovo electroporation is a technique which allows genetic manipulation of the avian embryo. Common applications of this technique include functional analysis of genes and putative enhancer elements. This video demonstrates neural tube electroporation in HH 10 chick embryos. Injection technique and proper egg handling are discussed.

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology.  With the advent of ...

Biology

In Ovo Electroporation in the Chicken Auditory Brainstem

Electroporation is a method that introduces genes of interest into biologically relevant organisms like the chicken embryo. It is long established that the chicken embryo is an effective research model for studying basic biological functions of auditory system development. More recently, the chicken embryo has become particularly valuable in studying gene expression, regulation and function associated with hearing. In ovo electroporation can be used to target auditory brainstem regions responsible for highly specialized auditory functions. These regions include the chicken nucleus magnocellularis (NM) and nucleus laminaris (NL). NM and NL neurons arise from distinct precursors of rhombomeres 5 and 6 (R5/R6). Here, we present in ovo electroporation of plasmid-encoded genes to study gene-related properties in these regions. We show a method for spatial and temporal control of gene expression that promote either gain or loss of functional phenotypes. By targeting auditory neural progenitor regions associated with R5/R6, we show plasmid transfection in NM and NL. Temporal regulation of gene expression can be achieved by adopting a tet-on vector system. This is a drug inducible procedure that expresses the genes of interest in the presence of doxycycline (Dox). The in ovo electroporation technique &#8211; together with either biochemical, pharmacological, and or in vivo functional assays &#8211; provides an innovative approach to study auditory neuron development and associated pathophysiological phenomena.

In Ovo Electroporation in the Chicken Auditory Brainstem

Auditory brainstem neurons of avians and mammals are specialized for fast neural encoding, a fundamental process for normal hearing functions. These neurons arise from distinct precursors of embryonic hindbrain. We present techniques utilizing electroporation to express genes in the hindbrain of chicken embryos to study gene function during auditory development.

Electroporation is a method that introduces genes of interest into biologically relevant organisms like the chicken embryo. It is long established that ...

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain dopaminergic neurons is implicated in Parkinson's disease, Schizophrenia, depression, and dementia1-5. Thus, studying the regulation of midbrain dopaminergic neuron differentiation could not only provide important insight into mechanisms regulating midbrain development and neural progenitor fate specification, but also help develop new therapeutic strategies for treating a variety of human neurological disorders.
Dopaminergic neurons differentiate from neural progenitors lining the ventricular zone of embryonic ventral midbrain. The development of neural progenitors is controlled by gene expression programs6,7. Here we report techniques utilizing electroporation to express genes specifically in the midbrain of Hamburger Hamilton (HH) stage 11 (thirteen somites, 42 hours) chick embryos8,9. The external development of chick embryos allows for convenient experimental manipulations at specific embryonic stages, with the effects determined at later developmental time points10-13. Chick embryonic neural tubes earlier than HH stage 13 (nineteen somites, 48 hours) consist of multipotent neural progenitors that are capable of differentiating into distinct cell types of the nervous system. The pCAG vector, which contains both a CMV promoter and a chick &beta;-actin enhancer, allows for robust expression of Flag or other epitope-tagged constructs in embryonic chick neural tubes14. In this report, we emphasize special measures to achieve regionally restricted gene expression in embryonic midbrain dopaminergic neuron progenitors, including how to inject DNA constructs specifically into the embryonic midbrain region and how to pinpoint electroporation with small custom-made electrodes. Analyzing chick midbrain at later stages provides an excellent in vivo system for plasmid vector-mediated gain-of-function and loss-of-function studies of midbrain development. Modification of the experimental system may extend the assay to other parts of the nervous system for performing fate mapping analysis and for investigating the regulation of gene expression.

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

To assess the function and the regulation of genes during the development of midbrain dopaminergic neurons, we describe a method that involves in ovo electroporation of plasmid DNA constructs into embryonic chick ventral midbrain dopaminergic neuron progenitors. This technique can be used to achieve efficient expression of genes of interest to study different aspects of midbrain development and dopaminergic neuron differentiation.

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain ...

Neuroscience

Microinjection-based System for In Vivo Implantation of Embryonic Cardiomyocytes in the Avian Embryo

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a general challenge in developmental biology research. In the embryonic heart, this can be particularly difficult as regional differences in the expression of transcriptional regulators, paracrine/juxtacrine signaling cues, and hemodynamic force are all known to influence cardiomyocyte maturation. A simplified method to alter a developing cardiomyocyte's molecular and biomechanical microenvironment would, therefore, serve as a powerful technique to examine how local conditions influence cell fate and function. To address this, we have optimized a method to physically transplant juvenile cardiomyocytes into ectopic locations in the heart or the surrounding embryonic tissue. This allows us to examine how microenvironmental conditions influence cardiomyocyte fate transitions at single cell resolution within the intact embryo. Here, we describe a protocol in which embryonic myocytes can be isolated from a variety of cardiac sub-domains, dissociated, fluorescently labeled, and microinjected into host embryos with high precision. Cells can then be directly analyzed in situ using a variety of imaging and histological techniques. This protocol is a powerful alternative to traditional grafting experiments that can be prohibitively difficult in a moving tissue such as the heart. The general outline of this method can also be adapted to a variety of donor tissues and host environments, and its ease of use, low cost, and speed make it a potentially useful application for a variety of developmental studies.

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.

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a ...

Using Chicken Embryo as a Powerful Tool in Assessment of Developmental Cardiotoxicities

Chicken embryos are a classical model in developmental studies. During the development of chicken embryos, the time window of heart development is well-defined, and it is relatively easy to achieve precise and timely exposure via multiple methods. Moreover, the process of heart development in chicken embryos is similar to mammals, also resulting in a four-chambered heart, making it a valuable alternative model in the assessment of developmental cardiotoxicities. In our lab, the chicken embryo model is routinely used in the assessment of developmental cardiotoxicities following exposure to various environmental pollutants, including per- and polyfluoroalkyl substances (PFAS), particulate matter (PMs), diesel exhaust (DE) and nano materials. The exposure time can be freely selected based on the need, from the beginning of development (embryonic day 0, ED0) all the way to the day prior to hatch. The major exposure methods include air-cell injection, direct microinjection, and air-cell inhalation (originally developed in our lab), and the currently available endpoints include cardiac function (electrocardiography), morphology (histological assessments) and molecular biological assessments (immunohistochemistry, qRT-PCR, western blotting, etc.). Of course, the chicken embryo model has its own limitations, such as limited availability of antibodies. Nevertheless, with more laboratories starting to utilize this model, it can be used to make significant contributions to the study of developmental cardiotoxicities.

Chicken embryos, as a classical developmental model, are used in our lab to assess developmental cardiotoxicities following exposure to various environmental contaminants. Exposure methods and morphological/functional assessment methods established are described in this manuscript.

Chicken embryos are a classical model in developmental studies. During the development of chicken embryos, the time window of heart development is ...

Xenotransplantation of Human Stem Cells into the Chicken Embryo

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the behavior of cells in a standardized in vivo environment. The main advantages of the chicken embryo include low cost, high accessibility, ease of surgical manipulation and lack of a fully developed immune system. Xenotransplantation into chicken embryos can provide valuable information about cell proliferation, differentiation and behavior, the responses of cells to signals in defined embryonic tissue niches, and tumorigenic potential. Transplanting cells into chicken embryos can also be a step towards transplantation experiments in other animal models. Recently the chicken embryo has been used to evaluate the neurogenic potential of human stem and progenitor cells following implantation into neural anlage1-6. In this video we document the entire procedure for transplanting human stem cells into the developing central nervous system of the chicken embryo. The procedure starts with incubation of fertilized eggs until embryos of the desired age have developed. The eggshell is then opened, and the embryo contrasted by injecting dye between the embryo and the yolk. Small lesions are made in the neural tube using microsurgery, creating a regenerative site for cell deposition that promotes subsequent integration into the host tissue. We demonstrate injections of human stem cells into such lesions made in the part of the neural tube that forms the hindbrain and the spinal cord, and into the lumen of the part of the neural tube that forms the brain. Systemic injections into extraembryonic veins and arteries are also demonstrated as an alternative way to deliver cells to vascularized tissues including the central nervous system. Finally we show how to remove the embryo from the egg after several days of further development and how to dissect the spinal cord free for subsequent physiological, histological or biochemical analyses.

In this paper we present a method for transplanting human stem cells into various regions of the central nervous system of the chicken embryo. This provides an in vivo model for assessing the proliferation and differentiation of various types of human stem cells in embryonic tissue environments.

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the ...

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for discovering new therapeutic strategies. Advancements in imaging technology have enabled quantitative investigation of the organization and maturing of the body plan, but later stage embryonic morphogenesis is less clear. Chicken embryos are an attractive vertebrate animal model system for this application because of its ease of culture and surgical manipulation. Early embryos can be cultured for a short time on filter paper rings, which enables complete optical access for cell patterning and fate studies1,2. Studying advanced developmental processes such as cardiac morphogenesis are traditionally performed through a window of the eggshell3-5, but this technique limits optical access due to window size. We previously developed a simple method to culture whole embryos ex-ovo on hexagonal weigh boats for up to 10 days, which enabled high resolution imaging via ultrasonography6,7. These cultures were difficult to transport, limiting the types of imaging tools available for live experiments. We here present an improved shell-less culture system with a cost-effective, portable environmental chamber. Eggs were cracked onto a hammock created by a polyurethane membrane (cling wrap) affixed circumferentially to a plastic cup partially filled with sterile water. The dimensions of the circumference and depth of the hammock were both critical to maintain surface tension, while the mechanics of the hammock and water beneath helped dampen vibrations induced by transportation. A small footprint circulating water bath was also developed to enable continuous temperature control during experimentation. We demonstrate the ability to culture embryos in this way for at least 14 days without morphogenic defect or delay and employ this system in several microsurgical and imaging applications.

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

In this article, we present a simple methodology to enable long-term ex-ovo avian embryo culture. This technique is ideal for longitudinal experimentation requiring complete optical accessibility and/or sterile transportation in avian embryos.

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for ...

The Preparation of Chicken Ex Ovo Embryos and Chorioallantoic Membrane Vessels as In Vivo Model for Contrast-Enhanced Ultrasound Imaging and Microbubble-Mediated Drug Delivery Studies

The chicken embryo and the blood-vessel rich chorioallantoic membrane (CAM) is a valuable in vivo model to investigate biomedical processes, new ultrasound pulsing schemes, or novel transducers for contrast-enhanced ultrasound imaging and microbubble-mediated drug delivery. The reasons for this are the accessibility of the embryo and vessel network of the CAM as well as the low costs of the model. An important step to get access to the embryo and CAM vessels is to take the egg content out of the eggshell. In this protocol, three methods for taking the content out of the eggshell between day 5 and&#160;8 of incubation are described thus allowing the embryos to develop inside the eggshell up to these days. The described methods only require simple tools and equipment and yield a higher survival success rate of 90% for 5-day, 75% for 6-day, 50% for 7-day, and 60% for 8-day old incubated eggs in comparison to ex ovo cultured embryos (~50%). The protocol also describes how to inject cavitation nuclei, such as microbubbles, into the CAM vascular system, how to separate the membrane containing the embryo and CAM from the rest of the egg content for optically transparent studies, and how to use the chicken embryo and CAM in a variety of short-term ultrasound experiments. The in vivo&#160;chicken embryo and CAM model is extremely relevant to investigate novel imaging protocols, ultrasound contrast agents, and ultrasound pulsing schemes for contrast-enhanced ultrasound imaging, and to unravel the mechanisms of ultrasound-mediated drug delivery.

This protocol describes three methods on how to obtain and use 5 to 8-day old chicken embryos and their chorioallantoic membrane (CAM) as an in vivo&#160;model to study contrast-enhanced ultrasound imaging and microbubble-mediated drug delivery.

The chicken embryo and the blood-vessel rich chorioallantoic membrane (CAM) is a valuable in vivo model to investigate biomedical processes, new ...

Bioengineering

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

The chicken embryo provides an excellent model system for studying gene function and regulation during embryonic development. In ovo electroporation is a powerful method to over-express exogenous genes or down-regulate endogenous genes in vivo in chicken embryos1. Different structures such as DNA plasmids encoding genes2-4, small interfering RNA (siRNA) plasmids5, small synthetic RNA oligos6, and morpholino antisense oligonucleotides7 can be easily transfected into chicken embryos by electroporation. However, the application of in ovo electroporation is limited to embryos at early incubation stages (younger than stage HH20 - according to Hamburg and Hamilton)8 and there are some disadvantages for its application in embryos at later stages (older than stage HH22 - approximately 3.5 days of development). For example, the vitelline membrane at later stages is usually stuck to the shall membrane and opening a window in the shell causes rupture of the vessels, resulting in death of the embryos; older embryos are covered by vitelline and allantoic vessels, where it is difficult to access and manipulate the embryos; older embryos move vigorously and is difficult to control the orientation through a relatively small window in the shell.
In this protocol we demonstrate an ex ovo electroporation method for gene transfer into chicken embryos at late stages (older than stage HH22). For ex ovo electroporation, embryos are cultured in Petri dishes9 and the vitelline and allantoic vessels are widely spread. Under these conditions, the older chicken embryos are easily accessed and manipulated. Therefore, this method overcomes the disadvantages of in ovo electroporation applied to the older chicken embryos. Using this method, plasmids can be easily transfected into different parts of the older chicken embryos10-12.

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

A method of gene transfer into chicken embryos at later incubation stages (older than Hamburger and Hamilton stage (HH) 22) is described. This method overcomes disadvantages of in ovo electroporation applied to older chicken embryos and is a useful technique to study gene function and regulation at older developmental stages.

The chicken embryo provides an  excellent model system for studying gene function and regulation during  embryonic development. In ovo electroporation is ...

In ovo Electroporation of Chicken Embryos

Electroporation is a technique used in biomedical research that allows for the manipulation of gene expression via the delivery of foreign genetic material into cells. More specifically, in ovo electroporation is performed on early developing chicks (Gallus gallus domesticus) contained within their eggshells. In this procedure, DNA or knockdown constructs are first injected into a target tissue. However, the genetic material is unable to penetrate the plasma membrane to carry out its function within the cell. To solve this problem, an electrical field is applied, causing temporary disruptions to membrane stability. This electric field also causes the negatively charged nucleic acids to migrate toward the positively charged electrode through the holes in the plasma membrane, thus effectively driving the DNA or knockdown construct into the cell. The major advantage of this technique is that the delivery of genetic material can be localized to isolated cell types at specific developmental time points. As a result, the genetic mechanisms that govern individual developmental events can be examined.
This video provides an overview of the principles behind in ovo electroporation and introduces the tools required for the technique, including capillary needles, electrodes, and an electroporator. A step-by-step protocol for carrying out the procedure is also presented prior to discussion of a few fascinating examples of how the technique is used to perform a variety of genetic manipulations in chicken embryos.

In Ovo Intravascular Injection in Chicken Embryos

Summary

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An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

The Preparation of Chicken Ex Ovo Embryos and Chorioallantoic Membrane Vessels as In Vivo Model for Contrast-Enhanced Ultrasound Imaging and Microbubble-Mediated Drug Delivery Studies

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

In ovo Electroporation of Chicken Embryos