Neuroscience
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Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse
Summary January 18th, 2016
This protocol describes in detail how to specifically transfect different regions in the C57BL/6 central nervous system via in utero electroporation. Included in this protocol are detailed instructions for transfections of regions that develop into the cortex, hippocampus, thalamus, hypothalamus, lateral septal nucleus and striatum.
Transcript
The overall goal of this in vivo cell manipulation is to specifically modify cells of certain regions in the central nervous system of the mouse-like cortex, hippocampus, thalamus, hypothalamus, lateral septal nucleus, and striatum. This method can help to answer key questions concerning the development and function of the brain, for instance, through the identification of different signaling cascades and the determination of the level of cell differentiation. The main advantage of this technique is that it allows for fast and efficient manipulation of various cell types in the central nervous system without the time-consuming generation of genetically modified mice.
Generally, the demonstration of this method is helpful, because individuals new to this method will often struggle with finding the right electrode position, which is critical for specific electroporation. To begin this procedure, cut open the abdominal cavity of an anesthetized pregnant mouse with a scalpel. Prevent the opened abdominal cavity from drying by moistening it with 0.9%warm methyl alcohol saline solution.
Then, carefully extract the uterine horns using a pair of ring forceps. Next, position an E14 embryo so that there are no visible vessels above the puncture site. To have a better visualization, use a 5-millimeter electrode to locate the angle and position of the desired area in the brain for transfection.
For the in utero electroporation of cortical area, slowly inject 1.5 to two microliters of the DNA solution in the left lateral ventricle. And ensure that the insertion depth is one to two millimeters, measured from the uterine wall. Then, check for blue-green coloring with a sharp demarcation.
For better visualization, use a 5-millimeter electrode to locate the angle and position of the desired location in the brain for transfection. After that, place the center of the three-millimeter platinum electrode paddles just anterior of the ear primordia, and make sure that they are not placed above the vessels or the placenta. Subsequently, apply voltage to begin transfection.
For the in utero electroporation of hippocampal formation, inject two microliters of DNA solution in the right lateral ventricle of an E15 embryo. Then, place the negative pole of the three to five millimeter platinum electrode paddles just anterior of the ear primordium, and the positive pole at the middle of the ear primordium. Then, apply voltage to begin transfection.
For the in utero electroporation of the lateral septal nucleus and striatum, inject one microliter of DNA solution in the right lateral ventricle of an E12 embryo. Then, place the 5-millimeter electrodes at the level of the ear primordia. Subsequently, apply voltage to begin transfection.
For the in utero electroporation of the thalamus and hypothalamus, inject one to 1.5 microliters of DNA solution in the lateral ventricle of an E12 to E13 embryo. Then, wait for two to three minutes for the solution to diffuse into the third ventricle. Next, place the 5 or three-millimeter electrodes just posterior to the ear primordia.
After that, apply voltage to begin transfection. This schematic shows the appropriate positions for the positive and negative poles for transfecting cortical areas. And this one, shows the positions of the positive and negative pole for transfecting the hippocampal formations.
The positions of the positive and negative pole for transfecting the striatum and the lateral septal nucleus are shown here. And the appropriate positions of the electrodes for transfecting the thalamus and the hypothalamus are shown here. The specificity of transfection depends on the electrode size.
Increasing the size of electrode paddle reduces the specificity of the transfection. This is especially obvious in younger embryonic stages, but also visible in late embryonic stages. Once mastered, this technique can be done in 15 minutes if it is performed properly.
While attempting this procedure, it's important to handle the embryos and the mothers with great care. After watching this video, you should have a good understanding of how to transfect specific regions in the central nervous system of the Black 6 mouse.
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