The overall goal of this procedure is to target intern neurons in the developing mouse four brain by means of in utero electroporation. This is accomplished by first preparing the injection needles. The second step is to remove the embryonic chain from the anesthetized animal.
Then DNA injection and electroporation are performed in the brain. The final step is to allow the animal to recover from the surgery. Ultimately, in utero, electroporation is used to allow for selective targeting of inter neuron subtypes.
The advantage of these methods of our existing techniques, it's like nonspecific in utero electro operation, is that it provides the means to carry out cell autonomous analysis of a subset of cortical inter neurons. The high levels of GFP expression allow for the visualization and analysis of single inter. This method can help answer fundamental questions in the neurodevelopmental field, such as what are the rules governing the integration of maturing inter.
To begin this procedure, prepare the microinjection pipette by pulling a glass capillary with a pipette puller. Next, carefully remove the capillary and collect the bottom needle for microinjection. Now, place an anesthetized pregnant mouse ventral side up on the heating pad.
Cover its fates with a mask that will continue to supply isof fluorine throughout the surgery. Then apply eye lubricant to both eyes. Set the heating pad to 36 degrees Celsius to prevent hypothermia afterward.
Check for the absence of foot and tail reflexes by pinching the rear paws or the tail. Next, clean the skin on the abdomen with 70%ethanol. Use forceps to pull the skin upward.
Then make a two centimeter small vertical incision along the midline starting midway between the third and fourth pairs of mammary glands. Be careful not to damage the abdominal wall using round forceps. Gently pull the embryonic chain away from the cavity.
Let the chain rest on the wet wipes. Start by exposing one half of the uterus. Then continue to the electroporation of the embryos before moving on to the other half.
In this procedure, prepare DNA solution by dissolving the pellet obtained from a standard maxi prep purification protocol in distilled water. Then add fast green to the DNA solution at a ratio of one to 20 to allow visualization during injection. Next, cut the tip of a pre pulled micro pipette with fine forceps to leave six millimeters from the beginning of the shoulder to the end of the tip.
Afterward, load the micro pipette with one microliter of DNA solution with fast green for injection. Hold the head of the embryo with round tweezers. Insert the pipette in the brain at 45 degrees, aiming at the lateral ventricle located near the midline.
If the needle penetrates through the ventricle, slowly retract the pipette. The ventricle should turn green when the solution begins to fill it. At this point, stop moving the pipette and inject one microliter of DNA.
Then gently withdraw the pipette. Set the ECM eight 30 ator to 5 45 volt pulses of 50 milliseconds spaced with a 950 millisecond inter pulse interval. Dip the five millimeter paddle electrodes in PBS to ensure efficient current transmission.
Then place the electrode paddles around the embryo's head with a positive paddle on the ventricle containing the DNA solution. Place the positive paddle slightly lower than the negative one to create a ventral current through the ganglionic eminences. This will minimize ectopic expression in parametal cells.
Now deliver a pulse by pressing the foot pedal once, then remove the electrodes and wipe them clean. Repeat the steps for each of the embryos. After electro pering all of the embryos.
Pour three milliliters of PBS into the abdominal cavity and return the embryos to the abdominal cavity. Next, suture the abdominal wall with a curved needle and five oh silk suture, and then the skin. Apply lidocaine on the wound and administer 120 milligrams per kilogram of aspirin intitally for analgesia.
Remove the animal from the anesthesia tubing and allow recovery on the heating pad on a paper wipe. After that, return the animal to the cage. Keep it on the heating pad at 37 degrees Celsius and monitor the health status.
In this experiment, electroporated inter neurons are delineated by the expression of CGE E derived subtype markers. This image shows that the CGE derived inter neurons are electroporated with a DLX five six EGFP plasmid. This image shows a VIP expressing inter neuron at P eight.
The EGFP expression delineates the entire dendritic tree and axonal arbor of single neurons, and here is an NPY expressing inter neuron at P eight. The NP y expression delineates neuroglia form cells. Lastly, here is an NPY expressing inter neuron at P 15.
Once mastered, this technique can be done in 20 minutes if performed properly. By combining the use of specific plasmid and genetically modified mouse lines, it is possible to achieve temporal and special control in the expression of genes of interest. In some experiments, we use A-D-L-X-T-T-A plasmid to induce expression of AU dependent transgenes.
In these experiments, we silent expression of the transgene by administering doxycycline.
