The overall goal of this procedure is to identify novel candidate genes for brain malformation disorders and to directly confirm their causative role in-vivo. This strategy can help answer key questions in the field of neurobiology by discovering molecular pathways associated with brain development and correlated pathologies. The main advantage of this strategy is that it will help discovering novel genes associated with brain disorders and to develop new diagnostic tools.
Using this approach we could disclose to the crucial contribution of the C6orf70 gene in the pathogenesis of periventricular nodular heterotopia, a brain malformation caused by abnormal neuronal migration. Demonstrating the procedure will be Fabienne Schaller, an engineer from our laboratory. Prepare an E15 pregnant rat for the procedure as outlined in the written protocol and according to institutional and national guidelines.
Ensure that the animal is properly anesthetized, that ophthalmic ointment is applied to the eyes, and that the abdomen is properly shaved and disinfected. After using sharp scissors to make a two centimeter vertical incision in the skin and the muscle of the caudal abdomen midline carefully expose one uterine horn from the abdominal cavity. Apply 37 degree Celsius pre-warmed normal saline solution dropwise to moisturize the embryos.
Keep the uterus moist throughout the procedure. To inject DNA, gently hold an embryo in one hand with the head easily accessible. Then, using the other hand, carefully insert a glass capillary connected to a microinjector into the left lateral ventricle which is located one millimeter from the midline and at a depth of two to three millimeters.
Use the microinjector to inject approximately one microliter of DNA with fast green to allow visual monitoring of the injection. To electroporate the DNA into the cells, first drop normal saline solution onto the surface of the three by seven millimeter electrode. Then place the positive electrode on the injected side and the negative electrode on the uninjected right ventricle.
Use the foot controlled pedal to deliver five electrical pulse at 950 millisecond intervals. Following electroporation, return the uterine horn to the abdominal cavity and add sterile saline to allow the embryos to position more naturally and to account for intraoperative fluid loss. Close the muscle wall with absorbable surgical sutures, then the skin using a simple interrupted stitch.
Provide post-operative analgesia for up to 48 hours or as indicated by institutional guidelines. Return the rat to its clean home cage and monitor it until it emerges from anesthesia. After fixing the brains in 4%paraformaldehyde as outlined in the written portion of the protocol, wash in PBS for 10 minutes.
While washing, prepare 50 milliliters of 4%agar in PBS ensuring that the temperature of the dissolved agarose does not exceed 50 degrees Celsius. Pour the agarose into plastic embedding molds and then place the brains in the agarose. Place the embedded brains at four degrees Celsius and allow the agarose to polymerize for at least an hour.
After the agarose has polymerized trim excess agarose from around the brain, then glue the brain to the vibratome base plate with the anterior posterior access of the brain perpendicular to the blade. After waiting a few minutes for the glue to dry, place the plate with the glued brain into the vibratome chamber. Fill the vibratome with PBS and begin slicing 100 micron thick sections.
After sectioning, transfer the sections to microscope slides and remove excess liquid. Then add a few drops of mounting medium and place a cover slip on top of the sections. Allow to dry overnight.
Image the sections using 10x magnification on a laser scanning confocal microscope. To analyze the images, first convert the image in 8-bit using appropriate software for quantitative image analysis. Then select one representative GFP positive florescent cell and manually define its shape by clicking on estimate and using the free hand selection tool to draw the border of the cell.
The software automatically retains the diameter and intensity threshold of the cell. Click on find cells to allow the software to localize transfected cells throughout the cortex. Then, if needed, manually remove false positives.
Divide the whole thickness of the cortex into eight areas of interest, normalized in individual sections, by clicking on region tool, selecting eight region stripes, where stripes one and eight correspond to the ventricular zone and to the top of the cortical plate respectively. Next, click on apply to allow the software to count the relative position of GFP transfected cells in the whole cortex. Finally, click on save quantification to export data in an Excel file for analysis.
Array CGH and whole exome sequencing identified C6orf70 as a candidate gene for developmental gene abnormalities. Quantitative PCR determined that this gene, along with PHF10 and DLL1 are expressed in rat cerebral cortex. The following images show representative neocortical coronal sections of E20 rat brains 5 days after electroporation with either green fluorescent protein construct alone or combined with shRNA targeting the C6orf70 coding sequence or with the relative ineffective mismatch construct.
Filament A knock down was used as control of impaired neuronal migration. In-utero electroporation with shRNA targeting the C6orf70 or Filament A induced arrest of GFP positive cells within the ventricular zone as indicated by the white arrows, whereas the cells expressing GFP alone, or in combination with the mismatch construct did not alter neuronal migration. While attempting this procedure it's important to take to mind which state of cortical genesis you want to analyze, either neuronal proliferation, migration, or maturation.
Don't forget that learning to perform in-utero electroporation in rodent embryonic brain requires a long training period, skills, and regular practice.
