In Ovo Intravascular Injection in Chicken Embryos

Summary

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.

Abstract

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.

Introduction

Chicken embryos have been widely used for centuries in developmental, immunological, pathological, and other biological applications^1,2,3. 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 albumen^5,6,7. 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 production^9,10,11.

In ovo intravascular injection in chicken embryos is a well-established and commonly used method^12,13,14. In this paper, we show a comprehensive description of this protocol including injection materials, sites, dosage, and representative results.

Protocol

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.

1. Collect fertilized eggs from a local vendor or institution, which can be stored at 16°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.
2. Scrub the eggshell softly and slowly with 75% ethanol before transferring the eggs to the incubator.
3. Place the eggs sideways, neatly on a rack in an incubator at 37.8 °C and 60% humidity. After 60 h of incubation (HH stage 17), embryos will develop visible blood vessels and the heart begins to beat¹⁵.
   ​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.

2. Preparation before injection

1. 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°) and sterilize the capillaries under UV light for 2 h.
2. Prepare exogenous materials such as plasmids, packaged lentiviruses, and PGCs.
   1. Gently mix the plasmid pEGFP-N1 (an enhanced GFP expressing plasmid, 1µg/µL) with liposome at a 1:3 ratio (m/v). Add water to obtain a final DNA concentration of 10 ng/µL. Let the DNA mixture sit at 37 °C for 20 min before injection.
   2. Thaw viruses on ice before injection. The titer of lentivirus used for injection should be over 5 x 10⁶ Tu/mL.
   3. Dilute parental or modified gonad PGCs (E4.5, HH stage 24) to 2,000-5,000 cells/µL.
      ​NOTE: Here we have shown the injection procedure using trypan blue.

3. Windowing

1. 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.
2. 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.

4. Injection

1. 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.
2. Fill the glass needle with the exogenous solution (plasmid, lentivirus, or PGCs) using a pipette slowly, avoiding air bubblesin the needle.
3. Aim the needle with exogenous solution at the blood vessel, with the needle parallel to the blood vessel being injected.
4. Gently insert the filled needle into the vessel and turn on the pump to eject the solution (usually the injected volume is ~1-5 µL) 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.
   1. 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.
5. After injection, remove the egg from the stereo microscope and drop 200 µL 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.
6. Label and place the injected egg back into the incubator and incubate until the required developmental stage or hatching.

Representative Results

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.

Discussion

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.

Materials

Name	Company	Catalog Number	Comments
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