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Genome editing technology greatly facilitates the genetic modification of various cells and animals. The common marmoset ( Callithrix jacchus ), a small non-human primate which exhibits high reproductive efficiency, is a widely used animal model in biomedical research. Developing genome editing techniques in the common marmoset will further enhance its utility. Here, we report the successful establishment of a knock-in (KI) method for marmoset embryonic stem cells (ESCs), which is based on the CRISPR-Cas9 system. The use of CRISPR-Cas9, mediated by homologous recombination (HR), enhanced the KI efficiency in marmoset ESCs. Furthermore, we succeeded in performing KI in early-stage marmoset embryos. In the course of the experiments, we found that HR in the marmoset ESCs is innately highly efficient. This suggested that the marmoset possesses a repair mechanism for DNA double-strand breaks. The current study will facilitate the generation of genetically modified marmosets and gene function analysis in the marmoset.

Currently, we can label the certain cells by transducing specific genes, called reporter genes, and distinguish them from other cells. For example, fluorescent protein such as green fluorescence protein (GFP) is commonly used for cell labeling. However, fluorescent protein is difficult to observe in living animals. We can observe the reporter signals of the luciferin-luciferase system from the outside of living animals using in vivo imaging systems, although the resolution of this system is low. Therefore, in this study, we examined the reporter genes, which allowed the MRI-mediated observation of labeled cells in living animals. As a preliminary stage of animal study, we transduced some groups of plasmids that coded the protein that could take and store metal ions to the cell culture, added metal ions solutions, and measured their T1 or T2 relaxation values. Finally, we specified the best reporter gene combination for MRI, which was the combination of transferrin receptor, DMT1, and Ferritin-M6A for T1WI, and Ferritin-M6A for T2WI.

Genetically modified nonhuman primates (NHP) are useful models for biomedical research. Gene editing technologies have enabled production of target-gene knock-out (KO) NHP models. Target-gene-KO/knock-in (KI) efficiency of CRISPR/Cas9 has not been extensively investigated in marmosets. In this study, optimum conditions for target gene modification efficacies of CRISPR/mRNA and CRISPR/nuclease in marmoset embryos were examined. CRISPR/nuclease was more effective than CRISPR/mRNA in avoiding mosaic genetic alteration. Furthermore, optimal conditions to generate KI marmoset embryos were investigated using CRISPR/Cas9 and 2 different lengths (36 nt and 100 nt) each of a sense or anti-sense single-strand oligonucleotide (ssODN). KIs were observed when CRISPR/nuclease and 36 nt sense or anti-sense ssODNs were injected into embryos. All embryos exhibited mosaic mutations with KI and KO, or imprecise KI, of c-kit . Although further improvement of KI strategies is required, these results indicated that CRISPR/Cas9 may be utilized to produce KO/KI marmosets via gene editing.

Mutations in the transcriptional regulator Mecp2 cause the severe X-linked neurodevelopmental disorder Rett syndrome (RTT). In this study, we investigate genes that function downstream of MeCP2 in cerebral cortex circuitry, and identify upregulation of Irak1 , a central component of the NF-κB pathway. We show that overexpression of Irak1 mimics the reduced dendritic complexity of Mecp2 -null cortical callosal projection neurons (CPN), and that NF-κB signalling is upregulated in the cortex with Mecp2 loss-of-function. Strikingly, we find that genetically reducing NF-κB signalling in Mecp2 -null mice not only ameliorates CPN dendritic complexity but also substantially extends their normally shortened lifespan, indicating broader roles for NF-κB signalling in RTT pathogenesis. These results provide new insight into both the fundamental neurobiology of RTT, and potential therapeutic strategies via NF-κB pathway modulation. Rett syndrome is a neurodevelopmental disorder caused by mutations in Mecp2 . Here the authors show that Mecp2 loss-of-function leads to upregulation of the NF-κB pathway, and that reducing NF-κB signalling ameliorates phenotypes of Mecp2 -null mice, thus offering a potential therapeutic strategy.

Ex vivo studies of the brain are often employed as experimental systems in neuroscience. In general, brains for ex vivo MRI studies are usually fixed with paraformaldehyde to preserve molecular structure and prevent tissue destruction during long-term storage. As a result, fixing brain tissue causes microstructural changes and a decrease in brain volume. Therefore, the purpose of this study was to investigate the regional effect of brain volume and microstructural changes on the restricted diffusion of water molecules in the common marmoset brain using in vivo and ex vivo brains from the same individual. We used 9.4T magnetic resonance imaging and also compared the T2-weighted images and diffusion-weighted imaging (DWI) data between in vivo and ex vivo brains to investigate changes in brain volume and diffusion of water molecules in 12 common marmosets. We compared fractional anisotropy, mean diffusivity, AD (axial diffusivity), and radial diffusivity values in white matter and gray matter between in vivo and ex vivo brains. We observed that AD showed the strongest correlation with regional volume changes in gray matter. The results showed a strong correlation between AD and changes in brain volume. By comparing the in vivo and ex vivo brains of the same individual, we identified significant correlations between the local effects of perfusion fixation on microstructural and volumetric changes of the brain and alterations in the restricted diffusion of water molecules within the brain. These findings provide valuable insights into the complex relationships between tissue fixation, brain structure, and water diffusion properties in the marmoset brain.

Postsynaptic proteins play crucial roles in synaptic function and plasticity. During brain development, alterations in synaptic number, shape, and stability occur, known as synapse maturation. However, the postsynaptic protein composition changes during development are not fully understood. Here, we show the trajectory of the postsynaptic proteome in developing male mice and common marmosets. Proteomic analysis of mice at 2, 3, 6, and 12 weeks of age shows that proteins involved in synaptogenesis are differentially expressed during this period. Analysis of published transcriptome datasets shows that the changes in postsynaptic protein composition in the mouse brain after 2 weeks of age correlate with gene expression changes. Proteomic analysis of marmosets at 0, 2, 3, 6, and 24 months of age show that the changes in the marmoset brain can be categorized into two parts: the first 2 months and after that. The changes observed in the first 2 months are similar to those in the mouse brain between 2 and 12 weeks of age. The changes observed in marmoset after 2 months old include differential expression of synaptogenesis-related molecules, which hardly overlap with that in mice. Our results provide a comprehensive proteomic resource that underlies developmental synapse maturation in rodents and primates. The proteomic changes that occur during synapse development are not fully understood. In this work, the authors characterise the postsynaptic proteome changes that occur during development in male mice and marmosets.

Animal models of brain function are critical for the study of human diseases and development of effective interventions. Resting-state network (RSN) analysis is a powerful tool for evaluating brain function and performing comparisons across animal species. Several studies have reported RSNs in the common marmoset ( Callithrix jacchus ; marmoset), a non-human primate. However, it is necessary to identify RSNs and evaluate commonality and inter-individual variance through analyses using a larger amount of data. In this study, we present marmoset RSNs detected using > 100,000 time-course image volumes of resting-state functional magnetic resonance imaging data with careful preprocessing. In addition, we extracted brain regions involved in the composition of these RSNs to understand the differences between humans and marmosets. We detected 16 RSNs in major marmosets, three of which were novel networks that have not been previously reported in marmosets. Since these RSNs possess the potential for use in the functional evaluation of neurodegenerative diseases, the data in this study will significantly contribute to the understanding of the functional effects of neurodegenerative diseases.

Oxygen attachment dissociation (OAD) is a tandem mass spectrometry (MS/MS) technique for annotating the positions of double bonds (C=C) in complex lipids. Although OAD has been used for untargeted lipidomics, its availability has been limited to the positive ion mode, requiring the independent use of a collision-induced dissociation (CID) method. In this study, we demonstrated the OAD MS/MS technique in the negative-ion mode for profiling phosphatidylserines, phosphatidylglycerols, phosphatidylinositols, and sulfatides, where the fragmentation mechanism remained consistent with that in the positive ion mode. Furthermore, we proposed optimal conditions for the simultaneous acquisition of CID- and OAD-specific fragment ions, termed OAciD, where oxygen atoms and hydroxy radicals facilitate C=C position-specific fragmentation, while residual water vapor induces cleavage of low-energy covalent bonds as observed in CID. Finally, theoretical fragment ions were implemented in MS-DIAL 5 to accelerate C=C position-resolved untargeted lipidomics. The OAciD methodology was used to illuminate brain region-specific marmoset lipidomes with C=C positional information, including the estimation of C=C positional isomer ratios. We also characterized the profiles of polyunsaturated fatty acid-containing lipids, finding that lipids containing omega-3 fatty acids were enriched in the cerebellum, whereas those containing omega-6 fatty acids were more abundant in the hippocampus and frontal lobe. Oxygen attachment dissociation (OAD) has been limited to positive-ion mode, hindering comprehensive lipidomics analysis. Here, the authors extend OAD to negative-ion mode, introducing OAciD for simultaneous CID and OAD fragment acquisition, enabling detailed C=C positional analysis and revealing brain region-specific lipidome variations, with implications for understanding lipid distribution in neurological contexts.

Magnetic resonance imaging (MRI) is a non-invasive neuroimaging technique that is useful for identifying normal developmental and aging processes and for data sharing. Marmosets have a relatively shorter life expectancy than other primates, including humans, because they grow and age faster. Therefore, the common marmoset model is effective in aging research. The current study investigated the aging process of the marmoset brain and provided an open MRI database of marmosets across a wide age range. The Brain/MINDS Marmoset Brain MRI Dataset contains brain MRI information from 216 marmosets ranging in age from 1 and 10 years. At the time of its release, it is the largest public dataset in the world. It also includes multi-contrast MRI images. In addition, 91 of 216 animals have corresponding high-resolution ex vivo MRI datasets. Our MRI database, available at the Brain/MINDS Data Portal, might help to understand the effects of various factors, such as age, sex, body size, and fixation, on the brain. It can also contribute to and accelerate brain science studies worldwide.

The postsynaptic density (PSD) is a protein condensate composed of ~1,000 proteins beneath the postsynaptic membrane of excitatory synapses. The number, shape, and plasticity of synapses are altered during development. However, the dynamics of synaptic protein composition across development have not been fully understood. Here we show alterations of PSD protein composition in mouse and primate brains during development. Proteins involved in synapse regulation are enriched in the differentially expressed (288 decreased and 267 increased) proteins on mouse PSD after a 2-week-old. We find that the changes in PSD protein abundance in mouse brains correlate with gene expression levels in postnatal mice and perinatal primates. This alteration of PSD composition is likely to be defective in the brains of mouse models or patients with autism spectrum disorder (ASD). Finally, we demonstrate that the brain of the common marmoset (Callithrix jacchus) changes PSD composition after the juvenile period. The alteration of PSD composition after 2-month-old is distinct from that observed in mice. Our results provide a comprehensive architecture of the remodeling of PSD composition across development, which may explain the molecular basics of synapse maturation and the pathology of psychiatric disorders, such as ASD. Competing Interest Statement The authors have declared no competing interest.