Cuy650 5

Plasmids expressing GFP, or the DISC1 765–835 peptide or the DISC1 765–835 L822Q peptide mixed with EGFP expressing plasmid pSUbGW) (~ 2μg/μl) were delivered to ventricular zone of embryo brain by in utero electroporation at E13.5 as previously described (Yoon et al., 2014 (link); Yoon et al., 2017b ). Briefly, DNA was injected using a beveled and calibrated micropipette with a ~10 μm opening at 15 psi, then five pulses (43 V, 50 ms in duration with a 950 ms interval) were delivered with tweezer electrodes (CUY650-5, Nepa Gene) by a CUY21SC electroporator (Nepa Gene). 50 mg/kg of EdU was injected to a mom 24 hours after electroporation and embryos were sacrificed and fixed with 4% PFA 6 hours after EdU injection. All animal procedures were performed in accordance with the protocol approved by the Institutional Animal Care and Use Committee.

Day 45 forebrain organoids were transferred into PBS solution in a 10 cm petri dish for electroporation. A mixture of 0.5 μl of plasmid DNA and 0.05% Fast green was injected into the lumen of neural tube structures in forebrain organoids using a calibrated micropipette (Yoon et al., 2017 ). About 3–4 locations on one side of each forebrain organoid were targeted by the injection. The DNA-injected side of the organoid was placed toward the positive electrode in the middle of 5 mm gap of electrode paddles (CUY650-5, Nepa Gene). Five pulses (40 V, 50 ms in duration with a 950 ms interval) were delivered by a square wave electroporator (CUY21SC, Nepa Gene). After electroporation, organoids were transferred back to the SpinΩ bioreactor for further culturing.

In utero electroporation was performed as described previously (Yoon et al., 2014 (link)). In brief, timed-pregnant CD1 mice (Charles River Laboratory) at E13.5 or E14.5 were anesthetized and the uterine horns were exposed and approximately 1 to 2 μl of plasmid DNA, 0.5 μg/μl pCAG-GFP (Addgene plasmid: 11150) and 2.5 μg/μl cFUGW plasmid with the control shRNA, or the shRNA against mouse Mettl3, Cnot1 and Cnot7, was injected manually into the lateral ventricles of embryos using a calibrated micropipette. Five pulses (40 V, 50 ms in duration with a 950 ms interval) were delivered across the uterus with two 5-mm electrode paddles (CUY650-5, Nepa Gene) positioned on either side of the head by a square wave electroporator (CUY21SC, Nepa Gene). After electroporation, the uterus was placed back in the abdominal cavity and the wound was sutured. Mouse embryos were analyzed at E17.5. For FlashTag of RGCs, 1 μl of 10 μM of a carboxyfluorescein succinimidyl ester (CellTrace CFSE, ThermoFisher) was injected into the lateral ventricle of the E17.5 embryos using a calibrated micropipette. Mouse embryos were collected 3 hr later, fixed with with 4% paraformaldehyde in PBS overnight at 4°C for analysis. All animal procedures were performed in accordance with the protocol approved by the Johns Hopkins Institutional Animal Care and Use Committee.

At day 45~47, organoids were transferred into Petri dishes containing 37°C PBS, and 3 μl of plasmids expressing GFP, orDISC1 765–835 peptide or DISC1 765–835 L822Q peptide in cFUGW vector mixed with EGFP expressing plasmid (pSUbGW) and 0.01% fast green diluted in sterile PBS was injected into 4–5 ventricle-like cavities of neural tube structures in forebrain organoids at 5 psi, using a beveled and calibrated micropipette with a ~20 μm diameter opening. Five pulses (43 V, 50 ms in duration with a 950 ms interval) were delivered with tweezer electrodes (CUY650–5, Nepa Gene) by a CUY21SC electroporator (Nepa Gene) as previously described (Yoon et al., 2017a ; Yoon et al., 2017b ). Electroporated organoids were transferred back to Spin3 bioreactor and cultured until fixation. 4 days after electroporation, organoids were pulsed with 10 μM EdU for 2 hr. The media was then replaced and organoids were washed 3 times with fresh media.