Hi, I am Mohammed Islam. I'm a graduate student in Dr.Lee's lab in the Department of Biomedical Engineering at Rutger University. Today I'm going to show you how to perform targeted retinal injection and innovo electroporation.
In chi embryo we'll be ing chicken embryonic retina at the stage of Embry day four, which is about 96 hours after inflammation. This technique is considered a novel method for the study of retinal development. This method specifically target retinal progenitor cells resulting in the ability to visualize all retinal cell type at the single cell level.
It can also be applied to gain or loss of function studies where a gene of interest can be targeted to a study, normal development or disease of the retina. X can be stored in a wine cooler at about 13 degrees cell CS for up to one week. If the temperature is too high, embryos will start to develop abnormally while lower temperature causes high mortality.
Once you are ready to incubate, take out the eggs from the wine cooler and set the eggs vertically with the larger end up. Keep the eggs in the room temperature for at least two hours before putting them in the 37.5 Gcel Yes incubator. Incubate the eggs for about 96 hours, which is about Embry Day four.
To obtain embryos that are at the hamburger and Hamilton stage 22 PP micropipet needles from pool glass capillary tubes, and break the tip under a dissecting microscope with the tweezers to get a tip opening about 0.1 micrometer in diameter and a two millimeter taper. Needles with larger tips have difficulty piercing the vitamin membrane. While the smaller tips have difficulty loading and delivering the DNA solution, attach the needle to a Hamilton gastric syringe mounted on a micro manipulator.
Use a small piece of master plex silicone tube for attaching the needle to the syringe. Since the connections are tied, so no need to add mineral oil To seal this attachment, mix two microliter of reporter plasmid DNA solution with a concentration ranging three to six microgram per microliter and 0.2 microliter of fast green on a piece of peripheral fast green dye will help to visualize the injection, slowly load the needle with the mixture to free the vital membrane from the inner membrane. Rotate the egg gently about 180 degrees and wait for a few minutes.
Then rotate it back to original position and set it for electroporation. Wipe the forceps and eggshell with 70%ethanol to avoid infection to the embryo. Make a small hole on the egg immediately above the air cell with a pair of forceps.
Be careful not to crack the eggshell. Remove small pieces one at a time to make a small window. Carefully remove the inner membrane using the Forceps without touching the vital membrane to prevent damage to the brain or the heart.
Position the needle contralateral to the main bundle of blood vessels entering the eye as indicated by the arrow and pointing towards the beak. Pierce through the vital membrane, sclera retina, and vitreous humor. By a sudden, my push of the needle, if the needle pierces through the other end of the eye, it should be all right unless it damages any major blood vein.
Slowly pull back the needle at the edge of the opening and place it almost tangent to the outer wall of the eyeball. Insert the needle into the subretinal space between Sclera and retina. Inject the DNA until you can visualize The green solution, filling the side of the eyeball and pushing the retina inwards by creating a bulge.
Here is another good example of a successful injection with formation of a bulge, and It is illustrated in this diagram. If your needle placing was not correct, Then you will see the DNA solution is spreading inside the vitreous humor filling up the middle of the eyeball. Also, if you damage the retina too much, then you'll see the DNA Solution is coming out of the eyeball.
Slowly remove the needle and immediately Place the electrodes in parallel inside the egg. After soaking in PBS, push down the electrodes to submerge into the amniotic fluid in a way so that the injected eye is located between the electrodes. Avoid touching any major blood vein or heart with the electrodes while placing them.
Negative electrodes should be at the injection site so that DNA can be transported from subretinal space into the retina towards positive electrode. Electrolyte the retina with five pulses of 15 volt for 50 milliseconds with 950 milliseconds intervals. Carefully remove the electrodes and seal the window of the egg with pieces of clear scotch tape label and date the injected egg before putting it back to the incubator.
Typically, it takes about three to five minutes to complete the whole process of electro operation. GFP expression can be seen as early as eight hours after electro operation. However, you may wait until the embryo reaches the desired state before harvesting the egg.
I hope this video was helpful for doing your own experiment. Thank you for watching.
