Begin by preparing a sufficient amount of electroporation mix for the control and gene of interest. Connect the Petri dish electrode chamber to the electroporator. Then using Microloader tips, fill microinjection needles with 8 microliters of each electroporation mix.
Cut the tips of the needles before use to achieve a stable flow using fine scissors. Ensure to remove only a small part of the tip as a blunt and wide tip can severely damage the organoids. Under a microscope, choose five cerebral organoids with smooth borders and visible ventricle-like structures.
Then move them to a 35-millimeter cell culture dish containing prewarmed DMEM/F-12 using a cut 1000 microliter pipette tip. Now, carefully insert the needle through the wall of a ventricle-like structure and infuse it with the electroporation mix until visibly filled. Do not apply excessive pressure on ventricle-like structures to avoid bursting.
Transfer one microinjected cerebral organoid to the Petri dish electrode chamber with a small amount of DMEM/F-12. Arrange the organoid so that the surfaces of the microinjected ventricle-like structures face toward the electrode connected to the positive pole of the electroporator. Now, electroporate the cerebral organoids one by one using five pulses of 80 volts for 50 milliseconds each with an interval of one second.
If the microinjection and electroporation were performed under non-sterile conditions, move the electroporated organoids under a laminar flow hood to a new 35-millimeter cell culture dish filled with prewarmed DMEM/F-12. Then culture the electroporated organoids in DM with vitamin A on an orbital shaker at 55 RPM in a humified atmosphere of 5%carbon dioxide and 95%air at 37 degrees Celsius. The next day, after electroporation, check the cerebral organoids for successful electroporation under a conventional inverted fluorescence microscope.
Transfer the electroporated organoids to a 15-milliliter conical centrifuge tube using a cut 1000 microliter pipette tip and remove excess medium. Add a sufficient amount of 4%paraformaldehyde in DPBS and incubate for 30 minutes at room temperature. At the end of the incubation, aspirate the paraformaldehyde.
Then add 5 milliliters of DPBS, shake gently, and aspirate the DPBS. Store the organoids in DPBS at 4 degrees Celsius until further use. Two days after electroporation, GFP-positive cells are almost exclusively localized in the ventricular zone and are positive for Pax6, indicating that these cells are apical progenitors or newborn basal progenitors.
After 10 days, the GFP-positive cells are localized in the basal regions. These cells are also positive for Pax6 or NeuN, which is indicative of basal progenitors or neurons, respectively. 3-D reconstruction of the electroporated cerebral organoids to obtain an impression of the 3-D distribution of the GFP-positive cells is shown.
