The overall goal of this procedure is to transfect plasma DNA into the hypothalamus of mouse embryos developing in utero in order to activate or repress the function of a gene of interest. This is accomplished by first anesthetizing the pregnant mouse and exposing the uterus through laparotomy. The second step is to inject the DNA solution into the third ventricle of the embryonic brain.
Next, the positive electrode is positioned inside the uterus between the placenta and embryonic head. The negative electrode is placed on the outer uterine surface and electric pulses are applied. The final step is to remove the transfected embryonic brains six days later.
Ultimately, fluorescence microscopy is used to analyze the phenotypical alterations of the transfected cells, number, shape, position, marker expression, et cetera. The main advantages of this technique over existing methods like viral transfection are first, it does not require special precautions, and as two facilities, second targeting of a specific region is possible. And third, with Ute electroporation, the screening of candidate genes is faster.
Generally, individuals new to this method will struggle because injecting the DNA solution into the third ventricle and positioning the needle electrode properly require advanced manual skew together with some experience. This method can help answer questions in the field of hypothalamic development, particularly those relevant to cell migration, as well as how individual regions and nucle are genetically specified. Begin this procedure by pulling some good quality glass micro pipettes as they are essential to reduce initial high abortion rate due to loss of amniotic fluid.
To prepare DNA dissolve the purified endotoxin free plasma DNA in PBS containing 0.1%fast green to a final concentration of one to two micrograms per microliter. The fast green will make the injected solution visible in the embryonic brain ventricle. Next, load 10 microliters of the DNA solution into the glass micro pipette.
The then connect the glass micro pipette to the injection system or a mouth pipette. In this step, prepare the surgical table with a heating pad and the surgical instruments Turn on the cold light sources to facilitate the visualization of the embryos. Then disinfect the surgical tools using a glass bead sterilizer.
Next, anesthetize a pregnant mouse with isof fluorine. After the mouse has been anesthetized, apply ointment to its eyes to prevent drying. Subsequently fit the mask on its face to maintain anesthesia continuously.
Now place the mouse on a heating pad with its belly up. Secure its body in place by fixing its four limbs with tape. Make sure that the mouse is completely anesthetized by pinching its tail for reflux.
Then shave the abdominal surface and disinfect it with iodine solution. Make a longitudinal incision about one to one and a half centimeters long on the abdominal skin. Afterward, cut the peritoneum.
Next place cotton gauze around the incision. Rinse the gauze with PBS. Expose one uterine horn by pulling it out carefully with blunt forceps.
Avoid pulling the myometrium or the uterus tightly. Since high pressure inside the uterus will increase the chances of embryo fluid loss upon injection resulting in abortion. Then place the uterine horn on a PBS rinsed gauze, and rinse it with PBS every few minutes to keep it moistened.
Now, under the microscope, hold the uterus in such a way that the brain ventricles can be visualized. Do not extensively reposition the embryo that lies in an unfavorable position. This only increases the chances of abortion.
Look at the embryo's head from the top to locate the gap where fissure between the left and right cortical hemispheres. The hemispheres are easy to distinguish, and the lateral ventricles inside them can usually be perceived as somewhat darker shapes. If an embryo is found to be perfectly oriented for DNA injection, it is possible to pierce the uterine wall and enter the third ventricle at once.
Hold the glass micro pipette with the DNA prepared earlier at 45 degrees to the uterine wall and puncture it at the rostral end of the interhemispheric fissure, penetrating to about a one millimeter depth. In this way, the tip of the glass micro pipette will enter the third ventricle of the brain and not the lateral ventricle. After word inject about one microliter of DNA solution into the third ventricle, green fluid will fill the ventricle in a good injection, repeat the same procedure with all the embryos of the same uterus horn.
This will allow some time for the DNA solution to mix evenly with the ventricular fluid and reach the entire neuro epithelium. Now switch the electro perter on and adjust the settings according to the embryonic age. Use the stainless steel needle electrode as the positive pole and the round flat electrode as the negative pole, select the embryos with the dorsal side up for electroporation.
Then pierce the uterine wall next to the embryo's head between the amniotic sac and the placenta by thrusting the tip of the needle electrode downwards through it. Then use the index finger of the other hand as a thrust block. About five millimeters of the electrode tip should be between the amniotic sac and the uterine wall at about the level of the midbrain.
As this is the targeting electrode, its position will determine which part of the hypothalamic neuro epithelium is most likely to get Transfected. Next, position the round flat electrode outside the uterus wall on the opposite side of the embryo's head with the other hand. Then gently squeeze the embryo between the electrodes.
Use the pedal switch to apply voltage. After that, slowly pull the needle electrode out of the uterus while holding back the uterus with the index finger of the other hand. If amniotic liquid is lost through the punctured wall, usually the embryo will undergo abortion.
After the injection and electroporation of all embryos, place the uterus back into the abdomen of the mouse very carefully and position them exactly as they were before. Then moisten the peritoneal cavity with saline before closing it. Suture the peritoneum with surgical cat gut, then suture the skin with a more resistant suture.
Disinfect the abdomen surface with iodine solution and inject a nonsteroid anti-inflammatory subcutaneously for pain relief. Next, remove the mother from the anesthetic machine and place it in a cage, which is heated by a heating pad. Monitor the mouse until it is completely recovered from the anesthesia and check it daily to ensure it is recovering from the procedure without any sign of infection or pain.
In this step, harvest the embryos or postnatals according to the desired day of analysis. Select the embryos that were injected with DNA and electroporated and dissect the brains under this stereo microscope. Then check for green fluorescent signal in the appropriate region.
To analyze the brain of the selected embryo, fix the tissue for a short time in 4%paraldehyde. Next, embed it in aros 4%or in gelatin albumin. Then section the brain on the vibrato.
These figures show the hypothalamus after in utero transfection of plasma DNA, carrying fluorescent reporter genes, GFP or CFP at E 12.5. This is a sagittal section showing a hypothalamus transfected from rostral to coddle, and here is the transverse section with the labeled cells at different levels along the media lateral axis. This sagittal section shows a specifically transfected mammillary body and labeling of its characteristic axonal tree, and on the electro parade side of this transverse section shows a labeled band corresponding to neurons born on E 12.5.
To analyze the sections closer. The horizontal sections parallel to the plane of the radial glial processes emanating from the mammillary recess were prepared. The ambi O neurons born from neuro epithelium transfected on day E 12.5 were identified on day E 18.5 by means of antibodies against GFP.
In this example, the antibody labeled a restricted group of MBO cells. This group was located between two unlabeled lateral and medial MBO areas corresponding to MBO neurons, born before and after E 12.5, and the co staining with an antibody against neston. In red shows the migration mode of individually labeled neurons on radial glial processes of the neuro epithelium Once mastered, this technique can be done in 30 minutes if it is performed properly.
After watching this video, you should have a good understanding of how to inject the DNA into the third ventricle and how to position the needle as positive electrode between embryo, head and placenta in order to efficiently target the hypothalamus. Following this procedure. Other methods like immunohistochemistry or inid hybridization can be performed in order to answer additional questions about the phenotype of the transfected neurons.
