Genetically modified mice
All experimental protocols and animal care and handling were approved by the Ethics Committee of Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine (XHEC-F-2024-086). The bicistronic expression vector px330-Cas9 (Addgene, 42230) containing the Cas9 coding region and the sg sequence (5′-ACATATGCTTCGGAGACTCA-3′) was directly used for microinjection. A 134 bp oligonucleotide acted as the donor ssDNA for intracytoplasmic DNA microinjection (134 bp oligonucleotide sequence, with the edited bases indicated in bold: 5′-GAGTTTGCCGACATAT CCAAAGAGGACCAGATTAAGAAA CTGCATGATTTGCTGGGGCCACACATGCTGTGGAGACTCAAGGCAGATGTCTTTAAGAACATGCCAGCCAAGACCGA GCTCATTGTTCGAGTGGA-3′). For zygote intracytoplasmic DNA microinjection, the mixture of px330-Cas9-sg plasmid (50 ng μl–1) and 134 bp ssDNA donor (100 ng μl–1) was diluted in RNAase-free water.
All mice were bred and maintained according to Shanghai Laboratory Animal Center Institutional Animal Regulations. Mice were housed in a specific pathogen-free animal facility at the Center for Excellence in Molecular Cell Science (CEMCS) with autoclaved food, bedding and water. Animals were maintained at room temperature (20–25 °C) at a humidity of 30–70% on a 12/12-h light–dark cycle (7:00–19:00). All mouse studies were carried out following the guidelines of the Institutional Animal Care and Use Committee (IACUC) at the CEMCS. B6D2F1 mice (female C57BL/6J × male DBA/2) aged 6–8 weeks were used for zygote collection. ICR mice, purchased from Beijing Vital River Laboratory Animal Technology, were used for pseudo-pregnant foster mother and vasectomized males. Mice were generated through standard mouse breeding procedures in the CEMCS animal facility The Chd3emh34 (endonuclease-mediated mutation-human 34 bp) mouse line was generated through homologous recombination using CRISPR–Cas9 technology. For the Chd3emh34 mouse line, a 34 bp mouse sequence (5′-ACTGGGGCCACATATGCTTCGGAGACTCAAGGCG-3′) was exchanged for the human sequence (5′-GCTGGGGCCACACATGCTGTGGAGACTCAAGGCA-3′); edited bases are highlighted in bold.
B6D2F1 female mice (8 weeks old) were injected with 7 IU (international units) of pregnant mare’s serum gonadotropin (PMSG). Subsequently, 7 IU of human chorionic gonadotropin (hCG) was injected over 46-48 h. Female mice were housed with B6D2F1 male mice overnight. Zygotes were collected from oviducts of B6D2F1 females 24 h after hCG injection using hyaluronidase (Sigma, H3884). The px330-Cas9-sg plasmid and 134 bp ssDNA donor was thoroughly mixed before injection. Using a micromanipulator (Olympus) and a FemtoJet microinjector (Eppendorf), the injection mixture was injected into zygotes. The injected zygotes were cultured in AA-KSOM (Millipore, MR-106-D) medium for 24 h in an incubator at 37 °C with 5% CO 2 until they developed into two-cell embryos.
The pseudo-pregnant foster mothers were prepared by mating oestrous ICR female mice with vasectomized male mice on the same day as the injection. The two-cell embryos were transferred into oviducts of 0.5 days post coitum (d.p.c.) recipient. Recipient mothers delivered pups at 19.5 d.p.c.
The neonatal mouse tissues were lysed with lysis buffer (100 mM Tris HCl (pH 7.8), 0.2% SDS, 5 mM EDTA, 200 mM NaCl and 100 μg ml–1 proteinase K) at 55 °C for more than 6 h, and then the mixture was boiled at 95 °C for 10 min to deactivate proteinase K. All mice were genotyped with specific primers (primer-F: 5′-TTAGCAACTTGGAGGGCTTC-3′; primer-R: 5′-TCTGCATGGGGCCTAGCTCC-3′). The Chd3R1025W mutation was verified by Sanger sequencing. The primer sequences used for mouse construction and genotyping are listed in Supplementary Table 7.
Primary mouse cortical neuron culture
Mouse cortical neurons were extracted from 14.5-day-old embryos of either sex. Cerebral cortices were dissected and digested with 20 U ml–1 papain (LS003126, Worthington) at 37 °C for 30 min, then cultured at 100,000 cells per cm2 on Lab-Tek II chamber slides (154941, Thermo Fisher Scientific) in 0.5 ml per well of Neurobasal medium (21103-049, Gibco) supplemented with 0.2% B27 (17504-044, Gibco) and 2 mM GlutaMAX (35050-061, Gibco). Lipid transfection using Lipofectamine 2000 (11668-019, Invitrogen) with 0.2 μg of each vector was performed after 24 h of cultivation following the manufacturer’s protocol. The medium was changed every 2 days. For morphological analyses, neurons were immunofluorescently stained at day 3 in vitro to assess axonal morphology and at day 7 in vitro to evaluate dendritic morphology. Confocal microscopy was used to acquire images, and the total axonal length, axonal neurite length, dendritic branch numbers and dendritic branch length were quantified using the Simple Neurite Tracer plugin in ImageJ. The mouse Chd3 shRNA sequence is presented in Supplementary Table 8.
HEK293T cell culture and preparation of protein samples
HEK293T cells were cultured to approximately 90% confluency in a culture dish (Corning) with DMEM (11965092, Gibco) and 10% FBS. Cells were transfected with the corresponding expression plasmids using Lipofectamine 2000 (11668-019, Invitrogen) according to the manufacturer’s instructions. After 72 h of incubation, the cells were washed with 1× PBS, collected via gentle pipetting and lysed in loading buffer. The lysates were heated at 100 °C for at least 30 min to ensure complete protein denaturation and stored at −20 °C. Proteins from cultured neuronal cells were extracted using the same protocol as for HEK293T cells.
... continue reading