The aim of this procedure is to use in utero, electroporation, to visualize and to genetically manipulate dendrites and spines in the mouse cerebral cortex and hippocampus. This is accomplished by first injecting the desired plasmid, DNA solution into the lateral ventricle of embryos in utero. Next, using different positions of the Palo electrodes.
According to the DNA injection, the cerebral cortex or the hippocampus is electroporated, then a desired postnatal or adult stages. Mice are perfused and brains are collected. Finally, the brains are sectioned with a vibram.
Ultimately, dendrites and spines can be visualized using confocal microscopy. The technique of unitary electroporation present several advantages compared to the methods to study non right and spine. Indeed, this technique allows first to study this aspect directly in vivo.
Moreover, in comparison to the generation of knockout mice in electro operation is a rapid approach to study the effect of gene suppression or gene expression and don white and spine, not only in specific cell structure, but also at a specific pon of development. Using inducible system in inter electro operation is also a useful tool to identify functional interaction between genes involved in don write and spine development. Indeed, in contrast to viruses, for example, it is tight for us to combine multiple HNA or multiple tons gene in the same population of cells To begin dilute DNA to the desired concentrations in water and add fast green dye to a final concentration of 0.05%Use a micro perpet puller to pull glass needles for injection.
Place a pregnant mouse in an anesthetic induction chamber using isof fluorine and open the oxygen up to two liters per minute. When the animal loses its writing reflex, transfer it to a pre-surgery mask. Place a drop of IGEL on each eye and use an electric razor to shave the hair of the abdomen.
Clean the shaved area once with chlorhexidine to collect flying hair. Transfer the animal to a second mask in the surgical area with its back on the heating pad. Begin the surgery when the petal reflex has been lost.
Put on a mask and sterile gloves and cover the animal with a sterile drape. Clean the shaved area at least three times with chlorhexidine. Use a scalpel to make a vertical incision along the midline through the skin.
Using scissors. Make a similar incision on the muscle of the abdomen along the linear elbow. Choosing the most accessible embryos.
Place ring forceps between two embryos and carefully pull the embryonic chain out out of the abdominal cavity. From this point forward, keep the embryos hydrated with sterile prewarm PBS. Using ring forceps.
Gently manipulate the position of the embryo inside the amniotic sack and stabilize the head between the rings. Squeeze gently to push the embryo up closer to the uterine wall. With the other hand, take the capillary holder and carefully insert the needle into the middle of the hemisphere to target the lateral ventricle.
Taking care to minimize movement of the needle at the surface of the uterine wall, and avoiding piercing blood vessels, press the pedal to inject around 0.5 microliters of green DNA solution. Place the electrodes on the sides of the embryo's head with the positive pedal on the same side as the injected ventricle for cortex electroporation, or on the opposite side of the injected ventricle, the hippocampus electroporation. Then apply five 30 volt electrical pulses of 50 millisecond duration at one second intervals.
Avoid applying current across the placenta as this will result in embryo death To increase chances of survival, inject and electro operate all the embryos in under 30 minutes when completed at PBS, the abdominal cavity, and use the ring forceps to replace the uterine horn in its original location. Use vicryl absorbable sutures to close the abdomen, wall and the skin. Place the animal in a recovery chamber until it wakes up.
Then transfer it into a cage on a heating pad 24 and 48 hours after surgery. Check the behavior of the mouse to assess pain, suffering, or distress, and weigh the animal to analyze brains. At postnatal stages, use a vibrator to section previously perfused and postfix brains mount the sections in aqua poly mount to image dendrites and spines.
This figure shows examples of electroporated cells in the cerebral cortex, the ca one and dentate gyrus of the hippocampus while type mice were electroporated at E 14.5, where the GFP construct and brains were harvested at postnatal day 14. By injecting a small volume of DNA solution, a few cells are labeled, which allows the visualization of the dendritic arborization of isolated GFP positive cells, as well as their spines at higher magnification. While attempting this procedure, it's important to remember to inject a small volume of DNA in order to target only few cells, and to be able to visualize and to quantify the don't write in the spine or the electro neuros.
