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 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.
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.
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's body, and the calcium in the hen'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.
2. Preparation before injection
3. Windowing
4. Injection
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 (Figure 2). Materials injected at two different sites were able to spread to the whole embryo, indicating the diffusion through blood circulation. The visualization of the blue color confirms the success of the injection.
The pEGFP-N1 vector was wrapped with PEI to achieve a concentration of 1 µg/µL, while the lentiviral vector pLVX-EGFP was wrapped with PEI and diluted after titration to achieve a virus concentration of 5 x 106 Tu/µL. The 2.5 day embryos (HH stage 14-15) were injected with wrapped pEGFP-N1 or pLVX-EGFP. At 4.5 days (HH stage 24), the embryos were observed using stereoscopic fluorescence microscopy. Green fluorescence was observed in the embryo indicating the vector expression after injection. The results show that encapsulated plasmids by liposomes (PEI) and packaged lentivirus were expressed in the chicken embryo 2 days post-injection (Figure 3). The plasmid and lentiviral vector injection results proved the exogenous gene expression in the embryo, implying the possible application in gene transfer.
The PGCs were isolated from the genital ridge of embryos (E4.5, HH stage 24) and cultured for purification as in the previous publication16. The injected PGCs were labeled with the red fluorescent protein (RFP). At E6.0 days (HH stage 28), the gonad of recipient embryos was isolated and observed. The results showed that the donor PGCs (red points) were able to effectively enter and colonize recipient's gonads (Figure 4).
Figure 1: The schematic of the intravascular injection. (A) The timeline of the intravascular injection; (B) Two injection sites indicated by black arrows, the head (1) and the dorsal aorta (2); (C) The process of the intravascular injection. Please click here to view a larger version of this figure.
Figure 2: Diffusion of the tracing dye in different injection sites. Top: dorsal aorta; Bottom: Head. Scale bar = 50 mm. Please click here to view a larger version of this figure.
Figure 3: Expression of GFP in embryos 2 days after lentiviral vector and encapsulated plasmid injection. Scale bar = 100 mm. Please click here to view a larger version of this figure.
Figure 4: Visualization of injected red fluorescence-labeled PGCs in the recipient chicken embryo. (E6.0, HH stage 28) gonad. Scale bar = 500 µm. Please click here to view a larger version of this figure.
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 PGCs to the recipient's embryo gonad, but also successfully produced the viable offspring with embryo gonad transplanted with induced PGCs, which implies the grand application of this method in the future20.
The critical step of the method is the injection. The exogenous materials must be directly injected into the blood vessels, not too deep or too shallow. Thus, it requires experience working with chicken embryos and high injection skills which could be easily obtained through practice. Compared to the air-cell injection, high injection efficacy and accuracy are more easily achieved using this method. In addition to the limitations of high cost and complicated steps, electrotransfection is not applied when a big window is made, as it may lead to a low survival rate.
As a method for gene manipulation in chicken embryos, the method also has some limitations. Around half of the eggs were dead during the incubation after injection with a survival rate of 40%-60%. Meanwhile, the delivery efficiency is another important factor to consider, especially for PGC injection (in our case, the efficiency of plasmid and lentivirus injection is ~50%-60%, and that of PGC injection is ~30%-40%). In the future, the protocol may be optimized and the survival rate may be increased considerably, further improving the application field of this method.
In conclusion,this protocol demonstrates that the in ovo chicken embryo intravascular injection is specific for exogenous materials (vectors or PGCs) to be transferred into the embryos. Moreover, this method can be easily learned and applied in many fields.
The authors have nothing to disclose.
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.
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 |