Cuy21sc electroporator

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.

All IUE experiments were performed and analysed by experimenters blind to genotype in mixed males and females. Timed-pregnant mice (E13.5) were obtained and deeply anaesthetized with isoflurane (1.5–3% in oxygen) for IUE surgery. Plasmid pCAG-GFP (11150, Addgene) (1–2 μg μl⁻¹) was microinjected into the lateral ventricles of E13.5 embryos to target layer 6 neurons of the cortex. The embryo was held through the uterus with platinum plate electrode tweezers (CUY650P3; Protech International) and electroporated (five, 50 ms pulses of 33 V with an interval of 950 ms; CUY21SC electroporator, NEPA GENE)^{42 (link)–46 (link)}. After the IUE surgery, buprenorphine (1 mg kg⁻¹ (body weight)) was administered into the wound to alleviate discomfort. At E15.5, pregnant dams were injected intraperitoneally with 150 mg kg⁻¹ (body weight) of BrdU. Two hours later, brains from electroporated pups were collected, fixed with paraformaldehyde overnight, and dehydrated with 30% sucrose. Tissue was cryosectioned at 20 μm. Fluorescent double-staining (GFP and BrdU) was performed as described below.