This experiment demonstrates in ovo electroporation of chick midbrain to study the effect of exogenous gene expression on midbrain development and dopaminergic neuron differentiation. First, inject the midbrain of hamburger Hamilton, stage 11 chick embryos with desired plasmid, DNA, to specifically target exogenous DNA into the developing midbrain vesicle. Then using custom made small electrodes, electroporated the midbrain vesicle to deliver the exogenous DNA specifically into the midbrain neural progenitors.
Next, harvest stage 23 embryos with good M cherry expression in the midbrain and generate optimal midbrain transverse sections. Ultimately, immuno staining analysis for dopaminergic neuron markers such as tyrosine hydroxylase can show the differentiation of neural progenitors into mature dopaminergic neurons induced by the expression of genes of interest. The main advantage of this technique over other existing methods of in ovo electroporation is that this protocol can achieve membrane specific electroporation to study mirin dopaminergic neuron differentiation.
Though this method can provide insight into mid-brain development, it can also be applied to other parts of the nervous system for performing fate mapping analysis, and for investigating the regulation of gene expression. In order to acquire hamburger Hamilton, stage 11 embryos, position each fertilized egg on its side and mark the top with a pencil dash. Then place the trays on a rocker in a prewarm humidified incubator.
Check the humidity and water level every 24 hours at 41 hours Post fertilization. Remove the eggs from the incubator and store at room temperature. Clean the surface of each egg with 70%ethanol and wipe down.
Now position two four centimeter pieces of scotch tape side by side on an egg to make a large rectangle over the pencil dash. Smooth the tape against the egg to frame the window with a 10 milliliter syringe attached to an 18 gauge needle. Pierce the egg shell angling the needle away from the yolk so as to not disturb or damage the embryo.
Draw five milliliters of albumin from the egg. Clean the needle with 70%ethanol. Dry the needle before piercing the next egg.
Enter settings for the flaming brown micro pipette puller. Then pull thin walled capillary tubes into injection needles with a P 10 pipette. Take up five to 10 microliters of plasmid solution into a long tip capillary pipette tip.
Then load the glass needle slowly in a continuous column absent of air bubbles. Place the loaded needle in the needle holder attached to the micro manipulator and micro injector. Now under a dissection microscope, using a dark background to better visualize the needle, trim the needle at the point where the green fluid stops with spring scissors.
Cut a two to two and a half centimeter diameter window from the previous needle hole. For drawing albumin. Rotate the egg in one hand while cutting.
Remember to clean off the scissors with a kim wipe saturated in 70%ethanol in between eggs. Proceed to view the chick embryo under a dissection microscope If necessary, visualize the embryo by injecting 0.22 micrometer filtered 20%India ink in PBS under the chick embryo. Position the embryo in parallel to the path of the needle with the head away from the needle.
Slowly pierce the neural tube with a 45 degree angle at the midbrain hind brainin junction. Start injecting with the pico spritzer micro injectors Duration set at 25 milliseconds. Adjust time depending on trim of needle fill only the neural vesicle representing midbrain with plasmid DNA containing fast green dye.
After retrieving the injection needle, place three drops of pen strep solution on the embryo. Verify the settings of the B-T-X-E-M-C eight 30 electro por for efficient electroporation. At the ventral midbrain position, the small custom made L-shaped platinum electrodes at the same horizontal level as the bottom of the neural tube, such that the embryonic midbrain is centered between the two electrodes.
Press the BTX foot switch once note the air bubbles generated around the electrodes. With each successful electroporation, carefully remove the electrodes from the egg and wipe the electrodes off. With a 70%ethanol covered chem wipe, place another three drops of pen strep solution on the embryo.
Now cover the window with a five centimeter by five centimeter piece of clear packaging tape. Without touching the egg contents seal the egg well place the eggs back into the incubator with turntables off. When the desired stage is achieved, harvest living embryos into a common PBS filled Petri dish for each condition.
Next, a fluorescent dissection microscope. Screen the harvested embryos for m cherry expression, transfer positive embryos into individual wells of a 24 well dish. They'll each well with one milliliter of freshly made 2%para formaldehyde and incubate for three hours at four degrees Celsius.
Wash the fixed embryos in PB S3 times place embryos in 15%sucrose until they sync upon equilibration. Repeat the equilibration in 30%sucrose, then embed each embryo in OCT orient embryos properly to generate optimal midbrain. Cross-sections for subsequent analyses.
Prepare 18 micron thick cryostat sections of the thick midbrain for downstream analysis in the embryonic chick midbrain in ovo electroporation assay hh. Stage 11 chick embryos are injected with a plasmid cocktail of the m cherry fluorescent marker and genes of interest and a fast green dyed tracker of the injection process. After midbrain electroporation, the embryos are further incubated and the m cherry positive embryos are harvested, fixed and embedded.
Then midbrain cryo sections are prepared. Samples are analyzed by immuno staining with midbrain markers such as tyrosine hydroxylase in successfully injected and electroporated chick embryos. M cherry expression is typically visible in the midbrain region after further development using m cherry expression as an injection marker.
Embryos exhibiting proper m cherry expression are then fixed, embedded, and oriented to prepare midbrain cryo sections. Here, the specification of dopaminergic fate in electroporated neural progenitors is examined by the colocalization of m cherry and the flag tagged gene with the dopaminergic neuron marker tyrosine hydroxylase. After watching this video, you should have a good understanding for how to regionally restrict gene expression to embryonic midbrain dopaminergic neuron progenitors, including how to inject DNA constructs specifically into the midbrain region, and how to pinpoint electroporation using small custom made electrodes Following this procedure.
Other methods such as RNI mediated lockdown or expression of enhancers linked to report genes can perform to study gene function and gene regulation in specific regions and cell types of the nervous system.
