Cuy21 electroporator

In utero electroporation was performed as described previously (23 (link)). Briefly, in utero electroporation to the dorsal neocortex was performed by injecting the DNA plasmid solution (5 mg/ml) plus 1% Fast Green using a glass capillary into the E14.5 ICR mice ventricle. DNA mixture was 2- to 3-fold higher than that of the EGFP plasmid, which was the electroporation marker. Electroporation was performed using a CUY-21 electroporator (NEPA GENE) and the following parameters: four 50-ms-long pulse separated by 950-ms-long intervals at 33 V.

A cDNA encoding the monomeric derivative of DsRed fluorescent protein was cloned into the piggybac cDNA expression vector with the PTS1 (serine-lysine-leucine) sequence^{72 (link)} using the In-Fusion HD Cloning Kit (TAKARA Bio Inc., Shiga, Japan).
The LC3-GFP cDNA was amplified from the pEGFP-LC3 plasmid (Addgene #21073) using the Tks Gflex DNA polymerase (TAKARA Bio Inc.) and cloned into the piggybac cDNA expression vector. PTS1-DsRed and pEGFP-LC3 was introduced using a CUY21 electroporator (NEPA Gene, Tokyo, Japan). Fluorescent images were captured using a BZ-X710 all-in-one fluorescent microscope (KEYENCE Corporation, Osaka, Japan) and analyzed integrated values of fluorescent brightness for each channel by an image analysis software BZ-X Analyzer Ver 1.3 (KEYENCE Corporation). To determine whether each fluorescent protein localized to peroxisomes and autophagosomes, a chemical activator of peroxisome proliferation, Wy14643^{30 (link)} and that of autophagy, Rapamycin was used^{73 (link)}. DsRed/GFP double-positive dots were counted as peroxisomes processed by autophagy (pexophagy).

Pregnant ICR mice were purchased from SLC Japan (Shizuoka, Japan). In utero electroporation was performed essentially as described previously [19] (link), [20] (link). Briefly, 2 μL of nucleotide solution containing expression plasmids and pSUPER-RNAi plasmid (2 μg each) was introduced into the lateral ventricles of embryos, followed by electroporation using a CUY21 electroporator (NEPA Gene, Chiba, Japan) with 50 ms of 30 V electronic pulses, six times with 950 ms intervals. It is most likely that the expression and RNAi plasmids were efficiently co-transfected into the majority of the neuronal progenitor/stem cells at the transfected region [19] (link). All electroporations were performed on embryonic day 14.5 (E14.5), and at least five brains were used for each experiment.

Neuronal transfections were performed by in-utero electroporation of E15 wild-type C57BL/6J mice as described previously [17] (link). Briefly, an E15 pregnant mother was anesthetized using 2% isoflurane and injected with 0.1 mg/kg of buprenorphrine as anesthetic. Embryonic pups within the intact uterus were temporarily removed from the abdomen and injected into the left hemisphere with 1 µl of DNA mixture, containing the appropriate CRISPR-construct and soluble GFP (10∶1), using a ∼50 µm-diameter pipette sharply beveled at 15°–20° (Narishige, Japan), visually confirming the proper site of correct injection by mixing 0.005% fast green with the DNA. To target transfection to the hippocampus, the head of the embryonic pup was placed between paddles of tweezer electrodes (CUY21 electroporator, NEPA GENE, Japan), with the positive terminal covering the lateral surface of the right hemisphere and the negative terminal covering the left hemisphere. Each injected embryo was then subjected to 5×50 ms/35 V electric pulses. Following electroporation, the intact uterus containing the pups was returned to the abdomen, and the mother’s abdomen sutured shut. Recordings were made from transfected pups 14–20 days following birth.

All the procedures for animal surgery and maintenance were performed following animal protocols approved by the Harvard Standing Committee on Animal Care and National Institutes of Health guidelines. To target neocortical layer 2/3 pyramidal neurons, E15.5 timed-pregnant female C57BL/6 mice (Charles River, Massachusetts, United States) were anesthetized by 2% isoflurane. Uterine horns were carefully exposed and 1∼2 μl of DNA at the concentration of 1 μg/μl was injected into the lateral ventricle of the left hemisphere of intrauterine embryos using a ∼30–50-μm-diameter pipette sharply beveled at 15°–20° (Narishige, Japan), visually confirming the proper site of correct injection by mixing 0.005% fast green mixed with the DNA. We used the same shRNA pLKO.1 vector that was used to create lentivirus that was used for all other assays and an eGFP-N1 expressing vector (Clontech) as negative control. After injection, the embryo head was held between the paddles of a forcep-type platinum electrode (0.5 mm diameter) and electric pulses were delivered five times per second (50 V, 50 ms) (CUY21 electroporator, NEPA GENE, Japan). Warm PBS was dropped onto embryos periodically to prevent drying. The uterus was placed back into the pregnant mouse, and the anterior muscle and the skin were sutured separately. Pups were housed with the dam until they were needed.