Whole Retinal Explants from Chicken Embryos for Electroporation and Chemical Reagent Treatments

The overall goal of this procedure is to dissect experimentally, manipulate and culture, whole retinal implants from chicken embryos. This is accomplished by first collecting both eyes from a chicken embryo and placing them in a Petri dish. The second step is to remove the scleral on loga and the pigment epithelium using fine forceps.

Next, the whole retinal explan is subjected to electroporation of plasmid DNA with flat circular electrodes. The final step is to incubate the retinal explan in culture medium. In a 24 well plate in an incubator.

Ultimately, the whole retinal implants are fixed, cryo sectioned, and analyzed by immunofluorescence microscopy to visualize the retinal neurons. The main advantage of this technique over existing methods, like in OVO electro operations, is that it is rapid, easy to perform and ensures reproducible results. This method can help answer key questions in the field of central nervous system development, such as how neurogenesis is affected by specific chemical reagents like enzyme inhibitors or gene products that target the cell cycle Following incubation.

Place eggs at the desired embryonic age into the laminar flow hood, and wipe the eggshells with 70%ethanol to avoid contamination of the embryo. Tap the side of the egg firmly against a hard surface to crack open the egg and then place the contents into a 100 millimeter Petri dish. Use a small spoon to transfer the embryo to a 35 millimeter Petri dish containing prewarm PBS at 37 degrees Celsius to prevent the tissue from drying.

Next, decapitate the embryo and place the 35 millimeter Petri dish containing the embryonic tissue onto a binocular stereo vision. Dissecting microscope in the laminar flow hood, use fine forceps to make an incision through the mouth ventral to the eye. Starting at the site of incision tear open the tissue surrounding the entire eye.

Next, pinch off the optic nerve and remove the intact eye from the eye socket. Collect both the left and right eye from the same embryo for use as either the control or the treated eye. Use a small spoon to transfer the eyes to a new 35 millimeter Petri dish containing prewarm PBS Using fine forceps.

Remove all the tissue around the eye, including the scleral on laga. Next, pinch a small incision in the pigment epithelium at the dorsal part of the eye, taking care not to damage the neural retina. Then gently tear open the pigment epithelium starting at the site of incision.

Leave the lens and the entire retina attached to the vitreous body. Remove the pigment epithelium, taking care along the ciliary body around the pupil in the anterior region of the eye, and along the choroid fissure as it is difficult to remove from these locations. Turn on the electro and set the voltage to 15 volts for a hamburger and Hamilton stage 20 to 25 retina or to 20 volts for a stage 26 to 27 retina.

Next, set the pulse repeats to five pulses of 50 milliseconds, pulse length with one second intervals. Use a pulse generator that can be controlled by a foot pedal on the floor as both hands will be needed to hold the electrodes in place. Connect two custom made paddle shaped platinum electrodes to the output socket on the pulse generator.

The electrodes shown here were made from 0.1 millimeter platinum plate rls and the 0.8 millimeter connecting wire was insulated. Using plastic tubing, carefully transfer the whole retinal explan to a vete using a polyethylene pasture pipette that has had the end cut off. Avoid using pipette tips as the retina tends to attach to the plastic and tear apart.

Next, use a 100 microliter pipette to gently remove all of the PBS surrounding the retinal explan. Take care not to touch the retina in order to avoid tearing it. Add 100 microliters of the diluted plasmid solution to the QVE and ensure that the whole retinal explan is covered with solution.

Gently push down the retinal explan with forceps if it floats to the top. Use forceps electrodes to position the lens to face any one side of the vete and direct the optic nerve to the bottom of the vete. Next, place the positive electrode in front of the lens and the negative electrode behind the retina without touching the tissue.

Apply the current by pressing down the foot pedal check for bubbles at the electrodes indicating a successful discharge. Use a 100 microliter pipette to remove all the plasmid mix from the qve without damaging the retina. The plasmid mix can be reused three to five times after removing the plasmid.

Fill up the vete with prewarm PBS. Use forceps to gently detach the retinal explan from the wall of the vete. Then use the polyethylene past your pipette to transfer the whole retinal explan into a 24 well plate containing one milliliter of prewarm culture.

Medium incubate only one retinal explan per well. Incubate the retinal explan at 37 degrees Celsius and 5%CO2 on a rotator shaker with a constant speed of 50 RPM. The rotation ensures maximum exposure to the medium and to prevent adhesion of the retina to the bottom of the 24 well plate.

After 24 hours of culturing, GFP positive cells were visible in a large part of the intact retina as electroporation resulted in a large retinal area taking up the plasmid and expressing the reporter gene. After sectioning single GFP positive cells were easily detected along the ACO basal axis of the retina. The whole retinal explan can be cultured for approximately 24 hours longer.

Incubation times may lead to developmental delay, morphological malformations, apoptosis, and eventually disintegration of the retina. A section from a whole retinal explan cultured for 24 hours was immuno stain with phospho histone three appearing here in the red channel phospho histone three label cells in late G two mase. The pH three positive cells were found on the apical side of the retina consistent with normal retinal development.

As shown here, this development can be blocked by the addition of a CDK one two inhibitor incubation with the inhibitor for four hours prior to imaging significantly reduced the number of pH three positive in the retina After its development. This technique has been successfully used to investigate reported gene expression from new DNA plasmid and to treat retinal explants with chemical reagents, including regulators of the DNA damage pathway and the cell cycle. Following this procedure, other methods like Innovo electro operations can be performed in order to further confirm the results.