The overall goal of this procedure is to perform gene transfer into older chicken embryos in vivo to study gene function and regulation at older developmental stages. This is accomplished by first cracking an egg at about incubation day 2.5 and transferring the entire embryo into a Petri dish system. The second step is to inject the plasmid, encoding the gene of interest into the appropriate part of the embryo.
After injecting the plasmid place electrodes beside the embryo and perform X ovo electroporation, the final step is to collect the electroporated embryos for analysis. Ultimately, immunohistochemistry is used to detect expression of transferred genes in the embryo. Today we will show you how to do the X oval electroporation using chicken BULs.
The main advantage of this technique is that X over electroporation can overcome the problems of in over electroporation used for gene transfer in older chicken embryos. Successful X ovo culture of chicken embryos requires the use of fresh fertilized eggs. Do not use eggs stored at 12 degrees Celsius for longer than one week.
Lay the eggs onto their long sides in a forced draft incubator at 37.5 degrees Celsius with 60%humidity after incubation for 2.5 days. When embryos are at about hamburger and Hamilton. Stage 17, take out the eggs from the incubator.
Label the top of each egg with a pencil to indicate the direction for cracking for each egg. Pour about 20 milliliters of sterile distilled water into a clean, large Petri dish with a diameter of 145 millimeters. Place a small sterile Petri dish with a diameter of 94 millimeters into the large Petri dish.
Next, crack each egg on the bottom against a sharp metal edge. Carefully open the egg and transfer its entire contents into the small Petri dish. To ensure the good quality of the embryo, the entire content of the egg should be kept in a normally round form in a Petri dish system without any injury to the vilin membrane.
After transferring the contents of each egg into the small dish, cover the large dish with a lid. Put the Petri dish systems into another incubator for further culture at 37.5 degrees Celsius with about 60%humidity prior to X ovo electroporation. Use a micro electrode polar to pull glass capillaries from glass tubes of 1.0 millimeter diameter.
Break the tip of the glass capillaries into an appropriate diameter. Next, set up the appropriate parameters for electroporation such as different voltages for the distinct tissue and size of the embryos. Connect the appropriate electrodes to the electro according to the target tissue and size of the embryos.
Prepare a plasmid solution containing plasmids, encoding a target gene and a marker gene. In this demonstration, cadherin seven or CAD seven is the target and green fluorescent protein or GFP is the marker. Add fast green to a final concentration of 0.1%to label the plasmid solution green.
Finally, use a mouth pipette to load the glass capillary with the plasmid solution. Chicken embryos from the X ovo culture that have been incubated for six days are used for X ovo electroporation. To begin this procedure, use fine forceps to carefully tear the tylene and amnion membranes over the optic tectum and add three drops of sterile 0.9%sodium chloride solution onto the surface of the tectum.
Using a mouth pipette, inject the plasmid solution from the glass capillary into the cavity of the tectum. Place the electrodes with a tungsten needle cathode and a rectangle plate anode beside the tectum. Immediately use the electro to apply electric pulses to the tectum.
Cover the large Petri dish with the lid and return it to the incubator two or three days after electroporation of plasmids encoding GFP and CAD seven into chicken tectum at E six, the tectum are collected and fixed for immuno staining. The results are shown in this figure at E eight. The GFP protein is strongly expressed in the tum as indicated by these whole mount images here.
Panel A is a bright field image. Panel B is A GFP image, and panel C is emerged. Image furthermore, in the sections of the tectum at E nine GFP protein visualized in green in Panel D and CAD seven protein visualized in red in panel E are co-expressed as represented by the yellow color in panel F.When done correctly, the efficiency of X ovo electroporation is 60 to 100%These results suggest that X ovo electroporation is a successful method for gene transfer into older chicken embryos in vivo.
After watching this video, you should have a good understanding of how to perform gene transfer into older chicken embryos and the good luck with your experiment.
