Loss-of-Function Approach in the Embryonic Chick Retina by Using Tol2 Transposon-Mediated Transgenic Expression of Artificial microRNAs

This methodology provides an loss-of-function approach for studies of retinal development in the chick. This technique supports rapid and persistent suppression of targets genes in a specific area of the retina. This approach can also be applied to other parts of the chick embryo, including both the neural and non-neural tissues.

For in ovo electroporation, prepare a 0.25%Fast Green solution by dissolving 25 milligrams of Fast Green FCF in 10 milliliters of PBS, then filter the solution using a 0.2 micron syringe filter. Next, prepare the DNA cocktail injection solution by mixing the microRNA emerald GFP plasmid with the Tol2 transposase expression plasmid at a two to one ratio. Then add the prepared Fast Green solution to the DNA cocktail at a one to 10 ratio for visualization of the injected area.

Next, set up the microinjection apparatus. The apparatus consists of a Hamilton syringe, an 18 gauge two inch needle, a two centimeter long polyvinyl chloride tubing, and a micropipette needle. Fill the Hamilton syringe with heavy mineral oil, then attach the 18 gauge needle to the syringe, and fill the inner space of the needle with oil by depressing the syringe plunger.

Next, attach the polyvinyl chloride tubing to the end of the needle, and fill the tubing with the oil. Attach the pulled micropipette needle to the tubing. Then under the dissecting microscope using fine forceps, break off the tip of the needle to a 10 to 20 micrometer diameter to make a small opening.

Fill the entire needle with oil. Next, put five microliters of the colored DNA cocktail onto a Petri dish. Under the dissecting microscope, place the tip of the micropipette needle into the DNA solution on the Petri dish, and slowly draw the solution into the needle.

After the pressure has equilibrated inside and outside the needle, take the tip of the needle out of the DNA solution, and keep it submerged in sterile PBS in a small beaker until injection. Then to set up the electroporation apparatus, set a pair of platinum electrodes firmly with an electrode holder on a micromanipulator. Adjust the spacing between the tip of the electrodes to two millimeters, and connect the electrodes to a square wave pulse generator with cables.

Next, for the DNA microinjection, remove a fertilized White Leghorn egg from the incubator and wipe the egg's surface with tissue paper soaked in 70%ethanol. Then attach an 18 gauge needle to a 10 milliliter syringe, and insert the needle through the blunt end of the egg, angled downward to avoid damaging the yolk. After withdrawing two to three milliliters of albumin from the egg, seal the hole with a piece of scotch tape, and candle the egg with light to ensure that the embryo and vitelline membrane are detached from the shell.

Next, using forceps, remove a piece of egg shell from the top of the egg, exposing the embryo. Do not window more than five eggs at any one time to prevent drying out of embryos during electroporation. Insert the tip of the needle into the optic vesicle from its proximal side at a 45 degree angle, and inject the DNA cocktail by slowly depressing the plunger until the blue colored solution fills the lumen.

Withdraw the needle, and place its tip back into PBS. For electroporation, set the pulse voltage to 15 volts, pulse length to 15 milliseconds, pulse interval to 915 milliseconds, and pulse number to five. After adding a few drops of HBSS onto the vitelline membrane over the embryo, use the micromanipulator to lower the electrodes into the HBSS perpendicular to the anterior-posterior axis of the embryo.

Place the electrodes on either side of the optic vesicle, ensuring that the electrodes do not touch the embryo or blood vessels, then apply pulsed electric fields. After the electroporation is complete, remove the electrodes, and gently clean the electrodes with a water soaked sterile cotton bud to avoid albumin accumulation. Seal the window with scotch tape, and reincubate the embryo until the desired developmental stage.

Transfection of individual pre-microRNA sequences into Nel AP expressing HEK293T cells resulted in significant suppression of Nel expression by constructs against nucleotides 482 to 502, 910 to 930, and 2461 to 2481. Chaining two microRNA sequences significantly enhanced the knockdown efficiency. All three combinations showed enhanced suppression activities compared to unchained individual pre-microRNA sequences.

Robust suppression was observed at least 13 days after transfection. When a Nel pre-microRNA construct targeting nucleotides 482 to 502 and 2461 to 2481 was co-transfected with the transposase expression vector into the chick retina, Nel expression in the retinal pigment epithelium was significantly reduced in emerald GFP expressing cells at embryonic day 4.5. In eight day old embryos, Nel expression also decreased in retinal ganglion cells.

In contrast, Nel expression in the retina was not affected by the introduction of control microRNA. Following this procedure, effects on target gene suppression can be examined by using various methods such as examinations of cell proliferation and differentiation, cell death, and cell and axon tracing.