Although pronuclear microinjection is the most widely used method for producing transgenic (Tg) animals, phenotypic characterization is usually performed from the next generation onwards because the production efficiency is limited. Conventional Cre-loxP-based conditional knockout (cKO) mouse production requires generating two genetically modified strains and multiple rounds of breeding before cKO mice are available for analysis. Here, we optimized a piggyBac transposon-based method of Tg mouse production and established conditions under which nearly all F0 embryos are Tg. Using a single-cell RNA sequencing-based strategy, we characterized mosaicism in F0 embryos and demonstrated that piggyBac-mediated transgene integration occurs early in embryonic development. We also achieved ∼70% efficiency in generating bacterial-artificial-chromosome-Tg mice. By combining this method with genome editing, we developed a strategy for tissue-specific-knockout phenotyping in the F0 generation. This platform expands experimental options for Tg animal production by supporting rapid F0-based phenotypic assessment and efficient founder generation for subsequent breeding.

Background/Objectives: CRISPR/Cas9-mediated genome editing enables efficient generation of knockout mouse models; however, frameshift mutations do not always result in complete loss of function. The factors influencing functional inactivation following frameshift mutations remain incompletely understood. Here, we tested whether frameshift-dominant targeting of exon 1 is sufficient to generate a null allele of Fgf10, a gene essential for limb formation. Methods: Guide RNAs (gRNAs) were selected using a machine learning-based pipeline to favor microhomology-mediated end joining (MMEJ)-dominant repair. Editing efficiency and indel profiles were assessed via amplicon sequencing in mouse embryonic stem cells (mESCs) and preimplantation embryos. Edited embryos were transferred to surrogate females and analyzed at embryonic day 15.5 (E15.5). Results: Amplicon sequencing confirmed >97% editing efficiency and >80% frameshift alleles in both mESCs and preimplantation embryos, with a predominant 7 bp deletion. Despite highly efficient frameshift-dominant editing, most of the E15.5 embryos were morphologically normal, indicating that exon 1 targeting did not reliably produce null phenotypes. In silico analysis suggested the possible presence of alternative downstream translation initiation sites, and our secretion assay supported this possibility. Initiation from a downstream ATG in a +2 reading frame (e.g., 7 bp deletion) may restore the downstream coding sequence and partially preserve protein function. Conclusions: Frameshift mutations in exon 1 of Fgf10 do not consistently result in functional knockout. The functional outcome depends on the specific reading frame and may be influenced by alternative translation initiation and protein domain architecture. These findings highlight important considerations for designing genome editing strategies to achieve complete gene inactivation.

Autosomal dominant polycystic kidney disease (ADPKD) is a common inherited disorder caused predominantly by heterozygous mutations in the PKD1 gene, leading to progressive renal cyst formation. While PKD1 mutant mouse models have provided mechanistic insights, PKD1 heterozygous mice fail to replicate the early cystogenesis observed in human patients. To address this gap, we conducted a longitudinal study using PKD1 heterozygous cynomolgus monkeys. Serial renal ultrasonography from birth to five years of age-corresponding to human childhood-revealed progressive cyst development. Remarkably, a subset of cysts, particularly smaller ones, exhibited spontaneous regression over time. This phenomenon was also observed in PKD1 mosaic monkeys harboring mixed variant patterns. In contrast, monkeys with biallelic PKD1 loss-of-function variants developed severe cystic disease, kidney enlargement, hepatic cysts, and elevated serum creatinine, resembling the clinical features of advanced ADPKD. These findings demonstrate that early renal cysts may possess intrinsic plasticity, challenging the conventional view of ADPKD as a relentlessly progressive disorder. Our results suggest that the early stages of cystogenesis represent a potential therapeutic window for intervention, in which cyst regression may be promoted. The PKD1 heterozygous monkey model thus provides a valuable platform for studying the developmental dynamics of ADPKD and for evaluating novel therapeutic approaches targeting early cystic changes.

Glisson's sheath is the connective tissue ensheathing the portal vein, hepatic artery, and bile duct within the liver. Although the connective tissue surrounding the liver surface is known as Glisson's capsule, its structure and function are poorly understood. In the present study, we analyzed mouse, rat, rabbit, feline, canine, monkey, porcine, bovine, and equine livers by histochemistry and immunohistochemistry analysis of α-smooth muscle actin, keratin 19, and podoplanin and examined how the structure of the Glisson's capsule is conserved and differs among the nine species. Glisson's capsule tended to thicken as the animal's body size increased. Among the nine mammalian species, bile ducts were observed adjacent to the connective tissue of Glisson's capsule in adult monkey, porcine, bovine, and equine livers without association with portal veins. Fetal monkey and porcine livers exhibited thin Glisson's capsules without bile duct development. The hepatic artery develops in the Glisson's capsule in adult canine, monkey, porcine, bovine, and equine livers without association with the bile ducts and portal veins. Similar to the human liver, the livers of adult monkeys develop lymphatic vessels beneath the liver surface. The present study reveals for the first time that the structure of the Glisson's capsule differs considerably between small (mouse, rat, rabbit, and cat) and large (monkey, pig, cattle, and horse) animals and that the dog exhibits an intermediate structure between the two groups.

Pronuclear microinjection is the most popular method for producing transgenic (Tg) animals. Because the production efficiency is typically less than 20%, phenotypic characterization of Tg animals is generally performed on the next generation (F1) onwards. However, apart from in rodents, in many animal species with long generation times, it is desirable to perform phenotyping in the founder (F0) generation. In this study, we attempted to optimize a method of Tg mouse production to achieve higher Tg production efficiency using piggyBac transposon systems and established optimal conditions under which almost all individuals in the F0 generation were Tg. We also succeeded in generating bacterial artificial chromosome Tg mice with efficiency of approximately 70%. By combining this method with genome editing technology, we established a new strategy to perform phenotyping of mice with tissue-specific knockout using the F0 generation. Taking the obtained findings together, by using this method, experimental research using Tg animals can be carried out more efficiently. ### Competing Interest Statement The authors have declared no competing interest.

Mislocalization and aggregate formation of TAR DNA-biding protein of 43kD (TDP-43) in the cytoplasm are signatures of amyotrophic lateral sclerosis(ALS) and frontotemporal lobar degeneration (FTLD). However, the role of two cytopathologies in ALS/FTLD pathogenesis is unclear. This study aims to elucidate the difference in their causality of TDP-43 in ALS/FTLD in vivo, using transgenic mice expressing human TDP-43 with defective nuclear localizing signals in neurons (Cyto-TDP) and those with aggregation propensity (Cyto-aggTDP). The expression levels of both proteins are less than half of endogenous TDP-43. Despite the low amount of Cyto-aggTDP, the TDP-43 phosphorylation is more evident than Cyto-TDP. Histopathological study showed accelerated astrogliosis in the anterior cerebral cortex of both mice. Cyto-aggTDP mice demonstrated significant but faint loss of neurons in the perirhinal(PERI) and ectorhinal(ECT) areas and higher Iba1-staining in the spinal cord than aged control. Despite the lack of locomotor dysfunctions in both mice, the open-field test showed enhanced exploratory behavior, indicating that the perpetual mislocalization of TDP-43 may suffice to trigger FTLD behavior. Besides, the aggregation propensity of TDP-43 promotes phosphorylation, but its role in the clinicopathological phenotype may not be primary.

Genetic mutations in fused in sarcoma (FUS) cause amyotrophic lateral sclerosis (ALS). Although mitochondrial dysfunction and stress granule have been crucially implicated in FUS proteinopathy, the molecular basis remains unclear. Here, we show that DHX30, a component of mitochondrial RNA granules required for mitochondrial ribosome assembly, interacts with FUS, and plays a crucial role in ALS-FUS. WT FUS did not affect mitochondrial localization of DHX30, but the mutant FUS lowered the signal of mitochondrial DHX30 and promoted the colocalization of cytosolic FUS aggregates and stress granule markers. The immunohistochemistry of the spinal cord from an ALS-FUS patient also confirmed the colocalization, and the immunoelectron microscope demonstrated decreased mitochondrial DHX30 signal in the spinal motor neurons. Subcellular fractionation by the detergent-solubility and density-gradient ultracentrifugation revealed that mutant FUS also promoted cytosolic mislocalization of DHX30 and aggregate formation. Interestingly, the mutant FUS disrupted the DHX30 conformation with aberrant disulfide formation, leading to impaired mitochondrial translation. Moreover, blue-native gel electrophoresis revealed an OXPHOS assembly defect caused by the FUS mutant, which was similar to that caused by DHX30 knockdown. Collectively, our study proposes DHX30 as a pivotal molecule in which disulfide-mediated conformational change mediates mitochondrial dysfunction and cytosolic aggregate formation in ALS-FUS.

DNA double-strand break (DSB) is the most severe form of DNA damage and accumulates with age, in which cytoskeletal proteins are polymerized to repair DSB in dividing cells. Since tau is a microtubule-associated protein, we investigate whether DSB is involved in tau pathologies in Alzheimer's disease (AD). First, immunohistochemistry reveals the frequent coexistence of DSB and phosphorylated tau in the cortex of AD patients. In vitro studies using primary mouse cortical neurons show that non-p-tau accumulates perinuclearly together with the tubulin after DSB induction with etoposide, followed by the accumulation of phosphorylated tau. Moreover, the knockdown of endogenous tau exacerbates DSB in neurons, suggesting the protective role of tau on DNA repair. Interestingly, synergistic exposure of neurons to microtubule disassembly and the DSB strikingly augments aberrant p-tau aggregation and apoptosis. These data suggest that DSB plays a pivotal role in AD-tau pathology and that the failure of DSB repair leads to tauopathy.

Aberrant angiogenesis is a pathological feature of a number of diseases and arises from the uncoordinated expression of angiogenic factors as response to different cellular stresses. Age-related macular degeneration (AMD), a leading cause of vision loss, can result from pathological angiogenesis. As a mutation in the mitochondrial ferritin (FTMT) gene has been associated with AMD, its possible role in modulating angiogenic factors and angiogenesis was investigated. FTMT is an iron-sequestering protein primarily expressed in metabolically active cells and tissues with high oxygen demand, including retina. In this study, we utilized the human retinal pigment epithelial cell line ARPE-19, both as undifferentiated and differentiated cells. The effects of proinflammatory cytokines, FTMT knockdown, and transient and stable overexpression of FTMT were investigated on expression of pro-angiogenic vascular endothelial growth factor (VEGF) and anti-angiogenic pigment epithelial-derived factor (PEDF). Proinflammatory cytokines induced FTMT and VEGF expression, while NF-κB inhibition significantly reduced FTMT expression. VEGF protein and mRNA expression were significantly increased in FTMT-silenced ARPE-19 cells. Using an in vitro angiogenesis assay with endothelial cells, we showed that conditioned media from FTMT-overexpressing cells had significant antiangiogenic effects. Collectively, our findings indicate that increased levels of FTMT inhibit angiogenesis, possibly by reducing levels of VEGF and increasing PEDF expression. The cellular models developed can be used to investigate if increased FTMT may be protective in angiogenic diseases, such as AMD.

Alzheimer's disease (AD) is the most common cause of dementia and understanding its pathogenesis should lead to improved therapeutic and diagnostic methods. Although several groups have developed transgenic mouse models overexpressing the human amyloid-I3 precursor protein (APP) gene with AD mutations, with and without presenilin mutations, as well as APP gene knock-in mouse models, these animals display amyloid pathology but do not show neurofibrillary tangles or neuronal loss. This presumably is due to differences between the etiology of the aged-related human disease and the mouse models. Here we report the generation of two transgenic cynomolgus monkeys overexpressing the human gene for APP with Swedish, Artic, and Iberian mutations, and demonstrated expression of gene tagged green fluorescent protein marker in the placenta, amnion, hair follicles, and peripheral blood. We believe that these nonhuman primate models will be very useful to study the pathogenesis of dementia and AD. However, generated Tg monkeys still have some limitations. We employed the CAG promoter, which will promote gene expression in a non-tissue specific manner. Moreover, we used transgenic models but not knock-in models. Thus, the inserted transgene destroys endogenous gene(s) and may affect the phenotype(s). Nevertheless, it will be of great interest to determine whether these Tg monkeys will develop tauopathy and neurodegeneration similar to human AD.

Aggregation of TAR DNA-binding protein of 43 kDa (TDP-43) is implicated in the pathogenesis of sporadic and certain familial forms of amyotrophic lateral sclerosis (ALS), suggesting elimination of TDP-43 aggregates as a possible therapeutic strategy. Here we generated and investigated a single-chain variable fragment (scFv) derived from the 3B12A monoclonal antibody (MAb) that recognises D247 of the TDP-43 nuclear export signal, an epitope masked in the physiological state. In transfected HEK293A cells, 3B12A scFv recapitulated the affinity of the full-length MAb to mislocalised TDP-43 with a defective nuclear localising signal and to a TDP-43 inclusion mimic with cysteine-to-serine substitution at RRM1. Moreover, 3B12A scFv accelerated proteasome-mediated degradation of aggregated TDP-43, likely due to an endogenous PEST-like proteolytic signal sequence in the VH domain CDR2 region. Addition of the chaperone-mediated autophagy (CMA)-related signal to 3B12A scFv induced HSP70 transcription, further enhancing TDP-43 aggregate clearance and cell viability. The 3B12A scFv also reduced TDP-43 aggregates in embryonic mouse brain following in utero electroporation while causing no overt postnatal brain pathology or developmental anomalies. These results suggest that a misfolding-specific intra body prone to synergistic proteolysis by proteasomal and autophagic pathways is a promising strategy for mitigation of TDP-43 proteinopathy in ALS.

The molecular machinery responsible for cytosolic accumulation of misfolded TDP-43 in amyotrophic lateral sclerosis (ALS) remains elusive. Here we identified a cullin-2 (CUL2) RING complex as a novel ubiquitin ligase for fragmented forms of TDP-43. The von Hippel Lindau protein (VHL), a substrate binding component of the complex, preferentially recognized misfolded TDP-43 at Glu246 in RNA-recognition motif 2. Recombinant full-length TDP-43 was structurally fragile and readily cleaved, suggesting that misfolded TDP-43 is cleared by VHL/CUL2 in a step-wise manner via fragmentation. Surprisingly, excess VHL stabilized and led to inclusion formation of TDP-43, as well as mutant SOD1, at the juxtanuclear protein quality control center. Moreover, TDP-43 knockdown elevated VHL expression in cultured cells, implying an aberrant interaction between VHL and mislocalized TDP-43 in ALS. Finally, cytoplasmic inclusions especially in oligodendrocytes in ALS spinal cords were immunoreactive to both phosphorylated TDP-43 and VHL. Thus, our results suggest that an imbalance in VHL and CUL2 may underlie oligodendrocyte dysfunction in ALS, and highlight CUL2 E3 ligase emerges as a novel therapeutic potential for ALS.

Pelizaeus-Merzbacher disease (PMD) is a hypomyelinating disorder caused by the duplication and missense mutations of the proteolipid protein 1 (PLP1) gene. PLP1 missense proteins accumulate in the endoplasmic reticulum (ER) of premature oligodendrocytes and induce severe ER stress followed by apoptosis of the cells. Here, we demonstrate that an anti-malaria drug, chloroquine, decreases the amount of an ER-resident mutant PLP1 containing an alanine-243 to valine (A243V) substitution, which induces severe PMD in human. By preventing mutant PLP1 translation through enhancing the phosphorylation of eukaryotic initiation factor 2 alpha, chloroquine ameliorated the ER stress induced by the mutant protein in HeLa cells. Chroloquine also attenuated ER stress in the primary oligodendrocytes obtained from myelin synthesis deficit (msd) mice, which carry the same PLP1 mutation. In the spinal cords of msd mice, chloroquine inhibited ER stress and upregulated the expression of marker genes of mature oligodendrocytes. Chloroquine-mediated attenuation of ER stress was observed in HeLa cells treated with tunicamycin, an N-glycosylation inhibitor, but not with thapsigargin, a sarco/ER Ca(2+)ATPase inhibitor, which confirms its efficacy against ER stress caused by nascent proteins. These findings indicate that chloroquine is an ER stress attenuator with potential use in treating PMD and possibly other ER stress-related diseases.

The cell-cycle transition from G1 to S phase has been difficult to visualize. We have harnessed antiphase oscillating proteins that mark cell-cycle transitions in order to develop genetically encoded fluorescent probes for this purpose. These probes effectively label individual G1 phase nuclei red and those in S/G2/M phases green. We were able to generate cultured cells and transgenic mice constitutively expressing the cell-cycle probes, in which every cell nucleus exhibits either red or green fluorescence. We performed time-lapse imaging to explore the spatiotemporal patterns of cell-cycle dynamics during the epithelial-mesenchymal transition of cultured cells, the migration and differentiation of neural progenitors in brain slices, and the development of tumors across blood vessels in live mice. These mice and cell lines will serve as model systems permitting unprecedented spatial and temporal resolution to help us better understand how the cell cycle is coordinated with various biological events.

Aggregation of TAR DNA-binding protein of 43 kDa (TDP-43) is a pathological signature of amyotrophic lateral sclerosis (ALS). Although accumulating evidence suggests the involvement of RNA recognition motifs (RRMs) in TDP-43 proteinopathy, it remains unclear how native TDP-43 is converted to pathogenic forms. To elucidate the role of homeostasis of RRM1 structure in ALS pathogenesis, conformations of RRM1 under high pressure were monitored by NMR. We first found that RRM1 was prone to aggregation and had three regions showing stable chemical shifts during misfolding. Moreover, mass spectrometric analysis of aggregated RRM1 revealed that one of the regions was located on protease-resistant β-strands containing two cysteines (Cys-173 and Cys-175), indicating that this region served as a core assembly interface in RRM1 aggregation. Although a fraction of RRM1 aggregates comprised disulfide-bonded oligomers, the substitution of cysteine(s) to serine(s) (C/S) resulted in unexpected acceleration of amyloid fibrils of RRM1 and disulfide-independent aggregate formation of full-length TDP-43. Notably, TDP-43 aggregates with RRM1-C/S required the C terminus, and replicated cytopathologies of ALS, including mislocalization, impaired RNA splicing, ubiquitination, phosphorylation, and motor neuron toxicity. Furthermore, RRM1-C/S accentuated inclusions of familial ALS-linked TDP-43 mutants in the C terminus. The relevance of RRM1-C/S-induced TDP-43 aggregates in ALS pathogenesis was verified by immunolabeling of inclusions of ALS patients and cultured cells overexpressing the RRM1-C/S TDP-43 with antibody targeting misfolding-relevant regions. Our results indicate that cysteines in RRM1 crucially govern the conformation of TDP-43, and aberrant self-assembly of RRM1 at amyloidogenic regions contributes to pathogenic conversion of TDP-43 in ALS.

Missense mutations in the proteolipid protein 1 (PLP1) gene cause a wide spectrum of hypomyelinating disorders, from mild spastic paraplegia type 2 to severe Pelizaeus-Merzbacher disease (PMD). Mutant PLP1 accumulates in the endoplasmic reticulum (ER) and induces ER stress. However, the link between the clinical severity of PMD and the cellular response induced by mutant PLP1 remains largely unknown. Accumulation of misfolded proteins in the ER generally leads to up-regulation of ER chaperones to alleviate ER stress. Here, we found that expression of the PLP1-A243V mutant, which causes severe disease, depletes some ER chaperones with a KDEL (Lys-Asp-Glu-Leu) motif, in HeLa cells, MO3.13 oligodendrocytic cells, and primary oligodendrocytes. The same PLP1 mutant also induces fragmentation of the Golgi apparatus (GA). These organelle changes are less prominent in cells with milder disease-associated PLP1 mutants. Similar changes are also observed in cells expressing another disease-causing gene that triggers ER stress, as well as in cells treated with brefeldin A, which induces ER stress and GA fragmentation by inhibiting GA to ER trafficking. We also found that mutant PLP1 disturbs localization of the KDEL receptor, which transports the chaperones with the KDEL motif from the GA to the ER. These data show that PLP1 mutants inhibit GA to ER trafficking, which reduces the supply of ER chaperones and induces GA fragmentation. We propose that depletion of ER chaperones and GA fragmentation induced by mutant misfolded proteins contribute to the pathogenesis of inherited ER stress-related diseases and affect the disease severity.

Accumulating evidence suggests that pathogenic TAR DNA-binding protein (TDP)-43 fragments contain a partial RNA-recognition motif domain 2 (RRM2) in amyotrophic lateral sclerosis (ALS)/frontotemporal lobar degeneration. However, the molecular basis for how this domain links to the conformation and function of TDP-43 is unclear. Previous crystal analyses have documented that the RRM2-DNA complex dimerizes under acidic and high salt conditions, mediated by the intermolecular hydrogen bonds of Glu246-Ile249 and Asp247-Asp247. The aims of this study were to investigate the roles of Glu246 and Asp247 in the molecular assembly of RRM2 under physiological conditions, and to evaluate their potential use as markers for TDP-43 misfolding due to the aberrantly exposed dimer interface. Unexpectedly, gel filtration analyses showed that, regardless of DNA interaction, the RRM2 domain remained as a stable monomer in phosphate-buffered saline. Studies using substitution mutants revealed that Glu246 and, especially, Asp247 played a crucial role in preserving the functional RRM2 monomers. Substitution to glycine at Glu246 or Asp247 induced the formation of fibrillar oligomers of RRM2 accompanied by the loss of DNA-binding affinity, which also affected the conformation and the RNA splicing function of full-length TDP-43. A novel monoclonal antibody against peptides containing Asp247 was found to react with TDP-43 inclusions of ALS patients and mislocalized cytosolic TDP-43 in cultured cells, but not with nuclear wild-type TDP-43. Our findings indicate that Glu246 and Asp247 play pivotal roles in the proper conformation and function of TDP-43. In particular, Asp247 should be studied as a molecular target with an aberrant conformation related to TDP-43 proteinopathy.

PLP1 amino acid substitutions cause accumulation of misfolded protein and induce endoplasmic reticulum (ER) stress, causing Pelizaeus–Merzbacher disease (PMD), a hypomyelinating disorder of the central nerve system. Currently no effective therapy is available for PMD. Promoted by its curative effects in other genetic disease models caused by similar molecular mechanisms, we tested if curcumin, a dietary compound, can rescue the lethal phenotype of a PMD mouse model (myelin synthesis deficient, msd). Curcumin was administered orally to myelin synthesis deficit (msd) mice at 180mg·kg−1·day−1 from the postnatal day 3. We evaluated general and motor status, changes in myelination and apoptosis of oligodendrocytes by neuropathological and biochemical examination, and transcription levels for ER-related molecules. We also examined the pharmacological effect of curcumin in cell culture system. Oral curcumin treatment resulted in 25% longer survival (p<0.01). In addition, oligodendrocytes undergoing apoptosis were reduced in number (p<0.05). However, no apparent improvement in motor function, neurological phenotype, and myelin formation was observed. Curcumin treatment did not change the expression of ER stress markers and subcellular localization of the mutant protein in vitro and/or in vivo. Curcumin partially mitigated the clinical and pathological phenotype of msd mice, although molecular mechanisms underlying this curative effect are yet undetermined. Nonetheless, curcumin may serve as a potential therapeutic compound for PMD caused by PLP1 point mutations.