RESUMEN
Direct delivery of genome-editing proteins into plant tissues could be useful in obtaining DNA-free genome-edited crops obviating the need for backcrossing to remove vector-derived DNA from the host genome as in the case of genetically modified organisms generated using DNA vector. Previously, we successfully delivered Cas9 ribonucleoprotein (RNP) into plant tissue by inserting microneedle array (MNA) physisorbed with Cas9 RNPs. Here, to enhance protein delivery and improve genome-editing efficiency, we introduced a bioactive polymer DMA/HPA/NHS modification to the MNA, which allowed strong bonding between the proteins and MNA. Compared with other modifying agents, this MNA modification resulted in better release of immobilized protein in a plant cytosol-mimicking environment. The delivery of Cas9 RNPs in Arabidopsis thaliana reporter plants was improved from 4 out of 17 leaf tissues when using unmodified MNAs to 9 out of 17 when using the polymer-modified MNAs. Further improvements in delivery efficiency can be envisaged by optimizing the polymer modification conditions, which could have significant implications for the development of more effective plant genome editing techniques.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Edición Génica/métodos , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Citosol/metabolismo , Preparaciones de Acción Retardada , ADN , Genoma de Planta/genéticaRESUMEN
The occurrence of accidental mutations or deletions caused by genome editing with CRISPR/Cas9 system remains a critical unsolved problem of the technology. Blocking excess or prolonged Cas9 activity in cells is considered as one means of solving this problem. Here, we report the development of an inhibitory DNA aptamer against Cas9 by means of in vitro selection (systematic evolution of ligands by exponential enrichment) and subsequent screening with an in vitro cleavage assay. The inhibitory aptamer could bind to Cas9 at low nanomolar affinity and partially form a duplex with CRISPR RNA, contributing to its inhibitory activity. We also demonstrated that improving the inhibitory aptamer with locked nucleic acids efficiently suppressed Cas9-directed genome editing in cells and reduced off-target genome editing. The findings presented here might enable the development of safer and controllable genome editing for biomedical research and gene therapy.
Asunto(s)
Aptámeros de Nucleótidos , Proteína 9 Asociada a CRISPR/antagonistas & inhibidores , Sistemas CRISPR-Cas , Edición Génica , Aptámeros de Nucleótidos/química , Células HEK293 , Humanos , Oligonucleótidos , ARN/antagonistas & inhibidores , Técnica SELEX de Producción de Aptámeros , Activación TranscripcionalRESUMEN
CRISPR-Cas9 technology has been at the forefront of the field of biology. The Streptococcus pyogenes (SpyCas9) protein forms a complex with guide RNA and can recognize and cleave double-stranded DNA through hybridization based on 20 base pairings. SpyCas9 has two nuclease domains, HNH and RuvC, each of which cuts each DNA strand, and both contain critical histidine residues. Although previously reported crystal structures provide useful geometric information, the extent to which these residues functionally contribute to catalysis is unknown. Here, we mutated histidine residues on HNH and RuvC domains to alanine or glycine and attempted to rescue the enzymatic activity by adding the imidazole molecule, using an in vitro DNA cleavage assay. H840A and H840G exhibited rescued enzymatic activity on the HNH domain following imidazole addition, suggesting that H840 acts as a general base. We also tested various chemicals and found that the pKa of imidazole derivatives, and not their molecular shape, correlated with the rescue effect. In contrast, both H983A and H983G on the RuvC domain did not exhibit a rescue effect following imidazole addition. Our chemical rescue approach will provide crucial insight into understanding Cas9 catalysis, complementing structural analyses.
Asunto(s)
Proteína 9 Asociada a CRISPR/química , Streptococcus pyogenes/enzimología , Sustitución de Aminoácidos , Proteína 9 Asociada a CRISPR/genética , ADN , Mutación Missense , Dominios Proteicos , Streptococcus pyogenes/genéticaRESUMEN
No specific and effective anti-viral treatment has been approved for COVID-19 so far. Systemic corticosteroid has been sometimes administered to severe infectious diseases combined with the specific treatment. However, as lack of the specific anti-SARS-CoV-2 drug, systemic steroid treatment has not been recommended for COVID-19. We report here three cases of the COVID-19 pneumonia successfully treated with ciclesonide inhalation. Rationale of the treatment is to mitigate the local inflammation with inhaled steroid that stays in the lung and to inhibit proliferation of the virus by antiviral activity. Larger and further studies are warranted to confirm the result of these cases.
Asunto(s)
Infecciones por Coronavirus/tratamiento farmacológico , Glucocorticoides/uso terapéutico , Neumonía Viral/tratamiento farmacológico , Pregnenodionas/uso terapéutico , Administración por Inhalación , Anciano , Betacoronavirus , COVID-19 , Femenino , Humanos , Pulmón/diagnóstico por imagen , Masculino , Pandemias , Radiografía Torácica , SARS-CoV-2 , Navíos , Tomografía Computarizada por Rayos X , Tratamiento Farmacológico de COVID-19RESUMEN
A 40-year-old man presented to the emergency department with periumbilical pain and fever. A computed tomographic scan confirmed multiple jejunal diverticulum with localized extraluminal air and panniculitis around it, and jejunal diverticulitis with localized perforation was suspected. His symptoms were mild, and extraluminal air was localized;therefore, he was advised bowel rest and administered only antibiotics. The patient's symptoms resolved without surgical treatment, and at the time of writing this report, there had been no recurrence. Small bowel diverticulitis is rare, and careful analysis of imaging studies is necessary for establishing a diagnosis. This was a rare case where small bowel diverticulitis was resolved without surgical treatment.
Asunto(s)
Diverticulitis , Divertículo , Perforación Intestinal , Enfermedades del Yeyuno , Adulto , Humanos , Intestino Delgado , Yeyuno , MasculinoRESUMEN
The CRISPR/Cas9 system is a powerful genome editing tool for disrupting the expression of specific genes in a variety of cells. However, the genome editing procedure using currently available vectors is laborious, and there is room for improvement to obtain knockout cells more efficiently. Therefore, we constructed a novel vector for high efficiency genome editing, named pGedit, which contains EGFP-Bsr as a selection marker, expression units of Cas9, and sgRNA without a terminator sequence of the U6 promoter. EGFP-Bsr is a fusion protein of EGFP and blasticidin S deaminase, and enables rapid selection and monitoring of transformants, as well as confirmation that the vector has not been integrated into the genome. By using pGedit, we targeted human ACTB, ACTG1 and mouse Nes genes coding for ß-actin, γ-actin and nestin, respectively. Knockout cell lines of each gene were easily and efficiently obtained in all three cases. In this report, we show that our novel vector, pGedit, significantly facilitates genome editing.
Asunto(s)
Actinas/antagonistas & inhibidores , Sistemas CRISPR-Cas , Edición Génica/métodos , Vectores Genéticos , Nestina/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/metabolismo , Actinas/genética , Aminohidrolasas/genética , Aminohidrolasas/metabolismo , Animales , Secuencia de Bases , Marcación de Gen , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Ratones , Nestina/genética , Regiones Promotoras Genéticas , Homología de SecuenciaRESUMEN
Osteoarthritis (OA), the most prevalent aging-related joint disease, is characterized by insufficient extracellular matrix synthesis and articular cartilage degradation, mediated by several proteinases, including Adamts-5. miR-140 is one of a very limited number of noncoding microRNAs (miRNAs) specifically expressed in cartilage; however, its role in development and/or tissue maintenance is largely uncharacterized. To examine miR-140 function in tissue development and homeostasis, we generated a mouse line through a targeted deletion of miR-140. miR-140(-/-) mice manifested a mild skeletal phenotype with a short stature, although the structure of the articular joint cartilage appeared grossly normal in 1-mo-old miR-140(-/-) mice. Interestingly, miR-140(-/-) mice showed age-related OA-like changes characterized by proteoglycan loss and fibrillation of articular cartilage. Conversely, transgenic (TG) mice overexpressing miR-140 in cartilage were resistant to antigen-induced arthritis. OA-like changes in miR-140-deficient mice can be attributed, in part, to elevated Adamts-5 expression, regulated directly by miR-140. We show that miR-140 regulates cartilage development and homeostasis, and its loss contributes to the development of age-related OA-like changes.
Asunto(s)
Cartílago/crecimiento & desarrollo , Homeostasis/fisiología , MicroARNs/genética , MicroARNs/metabolismo , Proteínas ADAM/metabolismo , Proteína ADAMTS5 , Animales , Desarrollo Óseo/genética , Homeostasis/genética , Articulación de la Rodilla/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Osteoartritis/patologíaRESUMEN
Skeletal muscles contain several subtypes of myofibers that differ in contractile and metabolic properties. Transcriptional control of fiber-type specification and adaptation has been intensively investigated over the past several decades. Recently, microRNA (miRNA)-mediated posttranscriptional gene regulation has attracted increasing attention. MiR-23a targets key molecules regulating contractile and metabolic properties of skeletal muscle, such as myosin heavy-chains and peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PGC-1α). In the present study, we analyzed the skeletal muscle phenotype of miR-23a transgenic (miR-23a Tg) mice to explore whether forced expression of miR-23a affects markers of mitochondrial content, muscle fiber composition, and muscle adaptations induced by 4 weeks of voluntary wheel running. When compared with wild-type mice, protein markers of mitochondrial content, including PGC-1α, and cytochrome c oxidase complex IV (COX IV), were significantly decreased in the slow soleus muscle, but not the fast plantaris muscle of miR-23a Tg mice. There was a decrease in type IId/x fibers only in the soleus muscle of the Tg mice. Following 4 weeks of voluntary wheel running, there was no difference in the endurance exercise capacity as well as in several muscle adaptive responses including an increase in muscle mass, capillary density, or the protein content of myosin heavy-chain IIa, PGC-1α, COX IV, and cytochrome c. These results show that miR-23a targets PGC-1α and regulates basal metabolic properties of slow but not fast twitch muscles. Elevated levels of miR-23a did not impact on whole body endurance capacity or exercise-induced muscle adaptations in the fast plantaris muscle.
Asunto(s)
Adaptación Fisiológica , MicroARNs/metabolismo , Fibras Musculares de Contracción Rápida/metabolismo , Fibras Musculares de Contracción Lenta/metabolismo , Esfuerzo Físico , Animales , Complejo IV de Transporte de Electrones/genética , Complejo IV de Transporte de Electrones/metabolismo , Femenino , Ratones , MicroARNs/genética , Fibras Musculares de Contracción Rápida/fisiología , Fibras Musculares de Contracción Lenta/fisiología , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Zinc-finger nucleases (ZFNs) are versatile reagents that have redefined genome engineering. Realizing the full potential of this technology requires the development of safe and effective methods for delivering ZFNs into cells. We demonstrate the intrinsic cell-penetrating capabilities of the standard ZFN architecture and show that direct delivery of ZFNs as proteins leads to efficient endogenous gene disruption in various mammalian cell types with minimal off-target effects.
Asunto(s)
Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Técnicas de Inactivación de Genes/métodos , Ingeniería Genética/métodos , Proteínas Recombinantes de Fusión/metabolismo , Dedos de Zinc/fisiología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Células CHO , Dominio Catalítico , Permeabilidad de la Membrana Celular , Cricetinae , Desoxirribonucleasas de Localización Especificada Tipo II/química , Humanos , Datos de Secuencia Molecular , Péptidos/química , Receptores CCR5/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Dedos de Zinc/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/químicaRESUMEN
MicroRNAs (miRNAs) have emerged as potential anticancer agents, but their clinical application is limited by the lack of an effective delivery system to tumors. Exosomes are small vesicles that play important roles in intercellular communication. Here, we show that synthetic miR-143 introduced into cells is released enveloped in exosomes and that the secreted exosome-formed miR-143 is transferred to osteosarcoma cells. The delivery of exosome-formed miR-143 significantly reduced the migration of osteosarcoma cells. The delivery efficiency of exosome-formed miR-143 was less than that achieved with lipofection, but the migratory potential of osteosarcoma cells was similarly inhibited after both strategies. Our results suggest that exosomes can deliver synthetic miR-143 and are a potentially efficient and functional delivery system.
Asunto(s)
Movimiento Celular , Exosomas/metabolismo , MicroARNs/administración & dosificación , Metástasis de la Neoplasia/prevención & control , Osteosarcoma/patología , Línea Celular Tumoral , Humanos , Células Madre Mesenquimatosas/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , MicroARNs/uso terapéutico , Metástasis de la Neoplasia/patología , Osteosarcoma/genética , Osteosarcoma/terapia , TransfecciónRESUMEN
Genome Editing is widely used in biomedical research and medicine. Zinc finger nucleases (ZFNs) are smaller in size than transcription activator-like effector (TALE) nucleases (TALENs) and CRISPR-Cas9. Therefore, ZFN-encoding DNAs can be easily packaged into a viral vector with limited cargo space, such as adeno-associated virus (AAV) vectors, for in vivo and clinical applications. ZFNs have great potential for translational research and clinical use. However, constructing functional ZFNs and improving their genome editing efficiency is extremely difficult. Here, the efficient construction of functional ZFNs and the improvement of their genome editing efficiency using AlphaFold, Coot, and Rosetta are described. Plasmids encoding ZFNs consisting of six fingers using publicly available zinc-finger resources are assembled. Two functional ZFNs from the ten ZFNs tested are successfully obtained. Furthermore, the engineering of ZFNs using AlphaFold, Coot, or Rosetta increases the efficiency of genome editing by 5%, demonstrating the effectiveness of engineering ZFNs based on structural modeling.
Asunto(s)
Edición Génica , Nucleasas con Dedos de Zinc , Edición Génica/métodos , Nucleasas con Dedos de Zinc/genética , Nucleasas con Dedos de Zinc/metabolismo , Humanos , Dedos de Zinc/genética , Sistemas CRISPR-Cas/genéticaRESUMEN
Background and Aim: To date, no randomized trials have compared the efficacy of 7-day vonoprazan, amoxicillin, and metronidazole triple therapy (VAM) versus 7-day vonoprazan, amoxicillin, and clarithromycin triple therapy (VAC) as a first-line treatment for Helicobacter pylori eradication. This study was performed to compare the efficacy of VAM and VAC as first-line treatments. Methods: This prospective multicenter randomized trial was performed in Japan and involved 124 H. pylori-positive patients without a history of eradication. Patients without antibiotic resistance testing of H. pylori were eligible. The patients were randomized to receive either VAC (vonoprazan 20 mg + amoxicillin 750 mg + clarithromycin 200 or 400 mg twice a day) or VAM (vonoprazan 20 mg + amoxicillin 750 mg + metronidazole 250 mg twice a day) for 7 days, with stratification by age and sex. Eradication success was evaluated using the 13C-urea breath test. We evaluated safety using patient questionnaires (UMIN000025773). Results: The intention-to-treat and per-protocol eradication rates of VAM were 91.3% (95% confidence interval [CI], 82.0-96.7%) and 92.6% (95% CI, 83.7-97.6%), respectively, and those of VAC were 89.1% (95% CI, 77.8-95.9%) and 96.1% (95% CI, 86.5-99.5%), respectively. No significant difference was observed between VAM and VAC in either analysis (P = 0.76 and P = 0.70, respectively). Abdominal fullness was more frequent in patients who received VAM than VAC. Conclusions: These findings suggest that VAM as a first-line treatment in Japan can be categorized as grade B (intention-to-treat cure rate of 90-95%) and have potential as a first-line national insurance -approved regimen.
RESUMEN
Cells release intraluminal vesicles in multivesicular bodies as exosomes to communicate with other cells. Although recent studies suggest an intimate link between exosome biogenesis and autophagy, the detailed mechanism is not fully understood. Here we employed comprehensive RNA interference screening for autophagy-related factors and discovered that Rubicon, a negative regulator of autophagy, is essential for exosome release. Rubicon recruits WIPI2d to endosomes to promote exosome biogenesis. Interactome analysis of WIPI2d identified the ESCRT components that are required for intraluminal vesicle formation. Notably, we found that Rubicon is required for an age-dependent increase of exosome release in mice. In addition, small RNA sequencing of serum exosomes revealed that Rubicon determines the fate of exosomal microRNAs associated with cellular senescence and longevity pathways. Taken together, our current results suggest that the Rubicon-WIPI axis functions as a key regulator of exosome biogenesis and is responsible for age-dependent changes in exosome quantity and quality.
RESUMEN
Sox9 plays a critical role in early chondrocyte initiation and promotion as well as repression of later maturation. Fellow Sox family members L-Sox5 and Sox6 also function as regulators of cartilage development by boosting Sox9 activation of chondrocyte-specific genes such as Col2a1 and Agc1; however, the regulatory mechanism and other target genes are largely unknown. MicroRNAs are a class of short, non-coding RNAs that act as negative regulators of gene expression by promoting target mRNA degradation and/or repressing translation. Analysis of genetically modified mice identified miR-140 as a cartilage-specific microRNA that could be a critical regulator of cartilage development and homeostasis. Recent findings suggest Sox9 promotes miR-140 expression, although the detailed mechanisms are not fully understood. In this study we demonstrate that the proximal upstream region of pri-miR-140 has chondrogenic promoter activity in vivo. We found an L-Sox5/Sox6/Sox9 (Sox trio) response element and detailed binding site in the promoter region. Furthermore, detailed analysis suggests the DNA binding and/or transactivation ability of Sox9 as a homodimer is boosted by L-Sox5 and Sox6. These findings provide new insight into cartilage-specific gene regulation by the Sox trio.
Asunto(s)
Cartílago/metabolismo , MicroARNs/metabolismo , Factor de Transcripción SOX9/metabolismo , Factores de Transcripción SOXD/metabolismo , Animales , Condrocitos/citología , Inmunoprecipitación de Cromatina , Dimerización , Regulación de la Expresión Génica , Células HEK293 , Proteínas del Grupo de Alta Movilidad/genética , Humanos , Ratones , Ratones Noqueados , Reacción en Cadena de la Polimerasa/métodos , Isoformas de Proteínas , Activación Transcripcional , TransgenesRESUMEN
MicroRNAs (miRNAs) are a class of noncoding small RNAs that act as negative regulators of gene expression. To identify miRNAs that may regulate human cell immortalization and carcinogenesis, we performed comparative miRNA array profiling of human normal and SV40-T antigen immortalized cells. We found that miR-296 was upregulated in immortalized cells that also had activation of telomerase. By an independent experiment on genomic analysis of cancer cells we found that chromosome region (20q13.32), where miR-296 is located, was amplified in 28/36 cell lines, and most of these showed enriched miR-296 expression. Overexpression of miR-296 in human cancer cells, with and without telomerase activity, had no effect on their telomerase function. Instead, it suppressed p53 function that is frequently downregulated during human cell immortalization and carcinogenesis. By monitoring the activity of a luciferase reporter connected to p53 and p21(WAF1) (p21) untranslated regions (UTRs), we demonstrate that miR-296 interacts with the p21-3'UTR, and the Hu binding site of p21-3'UTR was identified as a potential miR-296 target site. We demonstrate for the first time that miR-296 is frequently upregulated during immortalization of human cells and contributes to carcinogenesis by downregulation of p53-p21(WAF1) pathway.
Asunto(s)
Regiones no Traducidas 3' , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Regulación hacia Abajo , MicroARNs/metabolismo , Neoplasias/genética , Línea Celular , Línea Celular Tumoral , Transformación Celular Neoplásica , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/antagonistas & inhibidores , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Humanos , ARN Mensajero/metabolismo , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Regulación hacia ArribaRESUMEN
The Cre/lox recombination system has become a powerful technology for gene function analysis in a broad spectrum of cell types and organisms. In our previous report, Cre protein had been successfully delivered into intact Arabidopsis thaliana cells using electroporation. To expand the feasibility of the method of protein electroporation to other plant cells, here we attempt the protein electroporation into tobacco-derived BY-2 cells, one of the most frequently used plant cell lines for industrial production. In this study, we successfully deliver Cre protein into BY-2 cells with intact cell walls by electroporation with low toxicity. Targeted loxP sequences in the BY-2 genome are recombined significantly. These results provide useful information for genome engineering in diverse plant cells possessing various types of cell walls.
RESUMEN
MicroRNAs (miRNAs) are small noncoding RNAs that control essential cellular processes. For several decades, the molecular mechanisms underlying the functions and biogenesis of miRNAs have been clarified, whereas the molecular dynamics of miRNAs are poorly understood. We recently found that muscle-enriched miRNAs were reduced by only 20 ~ 50% in the skeletal muscles even 4 weeks after the suppression of miRNA processing through an inducible depletion of Dicer1 gene. These data suggest that miRNAs are stably expressed in skeletal muscle. In this study, we investigated the half-lives of those miRNAs in adult skeletal muscle with an in vivo metabolic labeling strategy and a genetic mouse model. In contrast to the hypothesis, in vivo metabolic labeling revealed that the half-lives of skeletal-muscle-enriched miRNAs were approximately 11-20 h. Furthermore, the levels of mature miR-23a decreased rapidly in the skeletal muscle of mice lacking miR-23 clusters in a tamoxifen-inducible manner. These data suggest that skeletal-muscle-enriched miRNAs are not highly stable in vivo. We also observed that the transfer of miR-150 into Dicer1-deficient muscle increased the miR-150 level to the same as that in control muscle. Taken together, our data demonstrate that miRNAs are degraded within a few days in adult skeletal muscle and that a Dicer-independent biogenetic pathway may produce mature miRNAs.
Asunto(s)
ARN Helicasas DEAD-box , MicroARNs , Músculo Esquelético , Animales , Ratones , MicroARNs/genética , MicroARNs/metabolismo , Músculo Esquelético/metabolismo , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Ribonucleasa III/genética , Ribonucleasa III/metabolismoRESUMEN
Osteoarthritis (OA) is the most common age-related joint disease. However, the role of many microRNAs (miRNA) in skeletal development and OA pathogenesis has not been sufficiently elucidated using genetically modified mice with gain- and loss-of-function models. We generated Cartilage-specific miR-26a overexpressing (Col2a1-Cre;miR-26a Tgfl/fl: Cart-miR-26a Tg) mice and global miR-26a knockout (miR-26a KO) mice. The purpose of the present study was to determine the role of miR-26a in OA pathogenesis using aging and surgically induced models. Skeletal development of Cart-miR-26a Tg and miR-26a KO mice was grossly normal. Knee joints were evaluated by histological grading systems. In surgically-induced OA and aging models (12 and 18 months of age), Cart-miR-26a Tg mice and miR-26a KO mice exhibited OA-like changes such as proteoglycan loss and cartilage fibrillation with no significant differences in OARSI score (damage of articular cartilage) compared with control mice. However, miR-26a KO mice reduced muscle strength and bone mineral density at 12 months of age. These findings indicated that miR-26a modulates bone loss and muscle strength but has no essential role in aging-related or post-traumatic OA.
Asunto(s)
Cartílago Articular , MicroARNs , Osteoartritis , Ratones , Animales , Osteoartritis/genética , Osteoartritis/patología , MicroARNs/genética , Ratones Noqueados , Debilidad Muscular , Condrocitos/patologíaRESUMEN
Muscle atrophy is caused by accelerated protein degradation and occurs in many pathological states. Two muscle-specific ubiquitin ligases, MAFbx/atrogin-1 and muscle RING-finger 1 (MuRF1), are prominently induced during muscle atrophy and mediate atrophy-associated protein degradation. Blocking the expression of these two ubiquitin ligases provides protection against muscle atrophy. Here we report that miR-23a suppresses the translation of both MAFbx/atrogin-1 and MuRF1 in a 3'-UTR-dependent manner. Ectopic expression of miR-23a is sufficient to protect muscles from atrophy in vitro and in vivo. Furthermore, miR-23a transgenic mice showed resistance against glucocorticoid-induced skeletal muscle atrophy. These data suggest that suppression of multiple regulators by a single miRNA can have significant consequences in adult tissues.
Asunto(s)
Regulación de la Expresión Génica , MicroARNs/metabolismo , Músculo Esquelético/metabolismo , Atrofia Muscular/metabolismo , Biosíntesis de Proteínas , Animales , Secuencia de Bases , Línea Celular , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Datos de Secuencia Molecular , Atrofia Muscular/genética , TransfecciónRESUMEN
Genome editing in plants employing recombinant DNA often results in the incorporation of foreign DNA into the host genome. The direct delivery of genome-editing proteins into plant tissues is desired to prevent undesirable genetic alterations. However, in most currently available methods, the point of entry of the genome-editing proteins cannot be controlled and time-consuming processes are required to select the successfully transferred samples. To overcome these limitations, we considered a novel microneedle array (MNA)-based delivery system, in which the needles are horizontally aligned from the substrate surface, giving it a comb-like configuration. We aimed to deliver genome-editing proteins directly into the inner layers of leaf tissues; palisade, the spongy and subepidermal L2 layers of the shoot apical meristem (SAM) which include cells that can differentiate into germlines. The array with needles 2 µm wide and 60 µm long was effective in inserting into Arabidopsis thaliana leaves and Glycine max (L.) Merr. (soybeans) SAM without the needles buckling or breaking. The setup was initially tested for the delivery of Cre recombinase into the leaves of the reporter plant A. thaliana by quantifying the GUS (ß-glucuronidase) expression that occurred by the recombination of the loxP sites. We observed GUS expression at every insertion. Additionally, direct delivery of Cas9 ribonucleoprotein (RNP) targeting the PDS11/18 gene in soybean SAM showed an 11 bp deletion in the Cas9 RNP target site. Therefore, this method effectively delivered genome-editing proteins into plant tissues with precise control over the point of entry.