In Ovo Intravascular Injection in Chicken Embryos

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.