Homology-directed repair-mediated knock-in (HDR-KI) in combination with CRISPR-Cas9-mediated double strand break (DSB) leads to high frequency of site-specific HDR-KI. While this characteristic is ...advantageous for generating genetically modified cellular and animal models, HDR-KI efficiency in mammalian cells remains low. Since avian DT40 cells offer distinct advantage of high HDR-KI efficiency, we expanded this practicality to adapt to mammalian research through sequential insertion of target sequences into mouse/human artificial chromosome vector (MAC/HAC). Here, we developed the simultaneous insertion of multiple fragments by HDR method termed the simHDR wherein a target sequence and selection markers could be loaded onto MAC simultaneously. Additionally, preparing each HDR donor containing homology arm by PCR could bypass the cloning steps of target sequence and selection markers. To confirm the functionality of the loaded HDR donors, we constructed a MAC with human leukocyte antigen A (HLA-A) gene in the DT40 cells, and verified the expression of this genomic region by reverse transcription PCR (RT-PCR) and western blotting. Collectively, the simHDR offers a rapid and convenient approach to generate genetically modified models for investigating gene functions, as well as understanding disease mechanisms and therapeutic interventions.
Mutations in leucine-rich repeat kinase 2 (LRRK2) have been identified as a genetic cause of familial Parkinson's disease (PD) and have also been found in the more common sporadic form of PD, thus ...positioning LRRK2 as important in the pathogenesis of PD. Biochemical studies of the disease-causing mutants of LRRK2 implicates an enhancement of kinase activity as the basis of neuronal toxicity and thus possibly the pathogenesis of PD due to LRRK2 mutations. Previously, a chemical library screen identified inhibitors of LRRK2 kinase activity. Here, two of these inhibitors, GW5074 and sorafenib, are shown to protect against G2019S LRRK2-induced neurodegeneration in vivo in Caenorhabditis elegans and in Drosophila. These findings indicate that increased kinase activity of LRRK2 is neurotoxic and that inhibition of LRRK2 activity can have a disease-modifying effect. This suggests that inhibition of LRRK2 holds promise as a treatment for PD.
We previously generated fully human antibody-producing TC-mAb mice for obtaining potential therapeutic monoclonal antibodies (mAbs). In this study, we investigated 377 clones of fully human mAbs ...against a tumor antigen, epithelial cell adhesion molecule (EpCAM), to determine their antigen binding properties. We revealed that a wide variety of mAbs against EpCAM can be obtained from TC-mAb mice by the combination of epitope mapping analysis of mAbs to EpCAM and native conformational recognition analysis. Analysis of 72 mAbs reacting with the native form of EpCAM indicated that the EpCL region (amino acids 24-80) is more antigenic than the EpRE region (81-265), consistent with numerous previous studies. To evaluate the potential of mAbs against antibody-drug conjugates, mAbs were directly labeled with DM1, a maytansine derivative, using an affinity peptide-based chemical conjugation (CCAP) method. The cytotoxicity of the conjugates against a human colon cancer cell line could be clearly detected with high-affinity as well as low-affinity mAbs by the CCAP method, suggesting the advantage of this method. Thus, this study demonstrated that TC-mAb mice can provide a wide variety of antibodies and revealed an effective way of identifying candidates for fully human ADC therapeutics.
Microcell-mediated chromosome transfer is an attractive technique for transferring chromosomes from donor cells to recipient cells and has enabled the generation of cell lines and humanized animal ...models that contain megabase-sized gene(s). However, improvements in chromosomal transfer efficiency are still needed to accelerate the production of these cells and animals. The chromosomal transfer protocol consists of micronucleation, microcell formation, and fusion of donor cells with recipient cells. We found that the combination of Taxol (paclitaxel) and reversine rather than the conventional reagent colcemid resulted in highly efficient micronucleation and substantially improved chromosomal transfer efficiency from Chinese hamster ovary donor cells to HT1080 and NIH3T3 recipient cells by up to 18.3- and 4.9-fold, respectively. Furthermore, chromosome transfer efficiency to human induced pluripotent stem cells, which rarely occurred with colcemid, was also clearly improved after Taxol and reversine treatment. These results might be related to Taxol increasing the number of spindle poles, leading to multinucleation and delaying mitosis, and reversine inducing mitotic slippage and decreasing the duration of mitosis. Here, we demonstrated that an alternative optimized protocol improved chromosome transfer efficiency into various cell lines. These data advance chromosomal engineering technology and the use of human artificial chromosomes in genetic and regenerative medical research.
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Uno and colleagues developed a novel protocol using Taxol and reversine to improve micronucleation and subsequent microcell-mediated chromosome transfer efficiency of mouse/human artificial chromosomes. Our results indicate induced spindle multipolarization and stimulated cell-cycle progression, providing the groundwork for the efficient transfer that advances genetic engineering and regenerative medicine research.
Neurodegenerative diseases represent an increasing burden in our aging society, yet the underlying metabolic factors influencing onset and progression remain poorly defined. The relationship between ...impaired IGF-1/insulin-like signaling (IIS) and lifespan extension represents an opportunity to investigate the interface of metabolism with age-associated neurodegeneration. Using data sets of established DAF-2/IIS-signaling components in Caenorhabditis elegans, we conducted systematic RNAi screens in worms to select for daf-2-associated genetic modifiers of α-synuclein misfolding and dopaminergic neurodegeneration, two clinical hallmarks of Parkinson’s disease. An outcome of this strategy was the identification of GPI-1/GPI, an enzyme in glucose metabolism, as a daf-2-regulated modifier that acts independent of the downstream cytoprotective transcription factor DAF-16/FOXO to modulate neuroprotection. Subsequent mechanistic analyses using Drosophila and mouse primary neuron cultures further validated the conserved nature of GPI neuroprotection from α-synuclein proteotoxicity. Collectively, these results support glucose metabolism as a conserved functional node at the intersection of proteostasis and neurodegeneration.
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•Insulin signaling modulates neurodegeneration in fly and worm Parkinson’s models•Reduced insulin-signaling screen reveals metabolic modifiers of protein misfolding•A glycolytic enzyme, GPI, is neuroprotective across worms, flies, and mouse neurons•GPI functions independently of DAF-16/FOXO to modulate proteostasis via glycolysis
Knight et al. screened for modifiers of α-synuclein misfolding and dopaminergic neurodegeneration, two clinical hallmarks of Parkinson’s disease, in worms with decreased IIS signaling. They identify the glycolytic enzyme GPI, which plays a conserved DAF-16/FOXO-independent role in worms, flies, and primary mouse neurons, integrating metabolic regulation with proteotoxicity and dopaminergic neurodegeneration.
α-synuclein (α-syn) is a main component of Lewy bodies (LB) that occur in many neurodegenerative diseases, including Parkinson's disease (PD), dementia with LB (DLB) and multi-system atrophy. α-syn ...mutations or amplifications are responsible for a subset of autosomal dominant familial PD cases, and overexpression causes neurodegeneration and motor disturbances in animals. To investigate mechanisms for α-syn accumulation and toxicity, we studied a mouse model of lysosomal enzyme cathepsin D (CD) deficiency, and found extensive accumulation of endogenous α-syn in neurons without overabundance of α-syn mRNA. In addition to impaired macroautophagy, CD deficiency reduced proteasome activity, suggesting an essential role for lysosomal CD function in regulating multiple proteolytic pathways that are important for α-syn metabolism. Conversely, CD overexpression reduces α-syn aggregation and is neuroprotective against α-syn overexpression-induced cell death in vitro. In a C. elegans model, CD deficiency exacerbates α-syn accumulation while its overexpression is protective against α-syn-induced dopaminergic neurodegeneration. Mutated CD with diminished enzymatic activity or overexpression of cathepsins B (CB) or L (CL) is not protective in the worm model, indicating a unique requirement for enzymatically active CD. Our data identify a conserved CD function in α-syn degradation and identify CD as a novel target for LB disease therapeutics.
Purpose: We explored a recovery correction technique that can correct metabolite loss during perchloric acid (PCA) extraction and minimize inter-assay variance in quantitative 1H nuclear magnetic ...resonance (NMR) spectroscopy of the brain and evaluated its efficacy in 5-fluorouracil (5-FU)- and saline-administered rats. Methods: We measured the recovery of creatine and dl -valine-2,3-d2 from PCA extract containing both compounds (0.5 to 8 mM). We intravenously administered either 5-FU for 4 days (total, 100 mg/kg body weight) or saline into 2 groups of 11 rats each. We subsequently performed PCA extraction of the whole brain on Day 9, externally adding 7 µmol of dl -valine-2,3-d2. We estimated metabolite concentrations using an NMR spectrometer with recovery correction, correcting metabolite concentrations based on the recovery factor of dl -valine-2,3-d2. For each metabolite concentration, we calculated the coefficient of variation (CEV) and compared differences between the 2 groups using unpaired t-test. Results: Equivalent recoveries of dl -valine-2,3-d2 (89.4 ± 3.9%) and creatine (89.7 ± 3.9%) in the PCA extract of the mixed solution indicated the suitability of dl -valine-2,3-d2 as an internal reference. In the rat study, recovery of dl -valine-2,3-d2 was 90.6 ± 9.2%. Nine major metabolite concentrations adjusted by recovery of dl -valine-2,3-d2 in saline-administered rats were comparable to data in the literature. CEVs of these metabolites were reduced from 10 to 17% before to 7 to 16% after correction. The significance of differences in alanine and taurine between the 5-FU- and saline-administered groups was determined only after recovery correction (0.75 ± 0.12 versus 0.86 ± 0.07 for alanine; 5.17 ± 0.59 versus 5.66 ± 0.42 for taurine µmol/g brain tissue; P < 0.05). Conclusion: A new recovery correction technique corrected metabolite loss during PCA extraction, minimized inter-assay variance in quantitative 1H NMR spectroscopy of brain tissue, and effectively detected inter-group differences in concentrations of brain metabolites between 5-FU- and saline-administered rats.
Radionuclide imaging and therapy are promising methods for controlling systemic cancers; however, their clinical application has been limited by excessive radionuclide accumulation in healthy ...tissues. To minimize radionuclide accumulation in non-cancerous tissues while ensuring sufficient build up in tumors, we aimed to develop a method that controlled the in vivo dynamics of radionuclides post-administration. To this end, we describe a novel strategy that combines liposomes, a potent carrier system for drug delivery, with unique radionuclide-ligand complexes based on 111In-ethylenedicysteine.
Conventional 111In-ligand-complexes-carrying liposomes delivered substantial amounts of radionuclides to tumors; however, they also accumulated in the liver and spleen. In contrast, 111In-ethylenedicysteine-carrying liposomes greatly reduced non-specific accumulation, while being retained selectively at high doses within tumors. Liposomes were rapidly broken down in the liver, releasing encapsulated 111In-ligand complexes. Among the chelates used, only 111In-ethylenedicysteine could escape from the liver and be excreted in the urine. Instead, most liposomes remained intact in tumors, retaining the radionuclide-ligand complexes within them. Therefore, high tumor accumulation was obtained regardless of the type of 111In-ligand complexes in the liposomes. In vivo single photon emission computed tomography/computed tomography imaging with 111In-ethylenedicysteine-carrying liposomes accurately revealed tumor-selective radionuclide retention with little background. Hence, our new strategy could greatly enhance tumor-to-healthy tissue ratios, improve diagnostic imaging, boost therapeutic efficacy, reduce toxicity to healthy tissues, and facilitate radionuclide imaging and therapy.
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•Novel strategy: liposomes+111In-ethylenedicysteine for precise radionuclide control.•Significantly reduced healthy tissue accumulation while preserving tumor targeting.•High tumor-to-healthy tissue ratio, precise imaging, and lower toxicity are achieved.•In vivo SPECT/CT imaging demonstrates tumor-selective radionuclide retention.•Potential for improved radionuclide imaging and radionuclide therapy for cancers.
Abstract
A ‘genomically’ humanized animal stably maintains and functionally expresses the genes on human chromosome fragment (hCF; <24 Mb) loaded onto mouse artificial chromosome (MAC); however, ...cloning of hCF onto the MAC (hCF-MAC) requires a complex process that involves multiple steps of chromosome engineering through various cells via chromosome transfer and Cre-loxP chromosome translocation. Here, we aimed to develop a strategy to rapidly construct the hCF-MAC by employing three alternative techniques: (i) application of human induced pluripotent stem cells (hiPSCs) as chromosome donors for microcell-mediated chromosome transfer (MMCT), (ii) combination of paclitaxel (PTX) and reversine (Rev) as micronucleation inducers and (iii) CRISPR/Cas9 genome editing for site-specific translocations. We achieved a direct transfer of human chromosome 6 or 21 as a model from hiPSCs as alternative human chromosome donors into CHO cells containing MAC. MMCT was performed with less toxicity through induction of micronucleation by PTX and Rev. Furthermore, chromosome translocation was induced by simultaneous cleavage between human chromosome and MAC by using CRISPR/Cas9, resulting in the generation of hCF-MAC containing CHO clones without Cre-loxP recombination and drug selection. Our strategy facilitates rapid chromosome cloning and also contributes to the functional genomic analyses of human chromosomes.
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