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1.
PLoS Biol ; 18(7): e3000747, 2020 07.
Artículo en Inglés | MEDLINE | ID: mdl-32644995

RESUMEN

CRISPR-Staphylococcus aureus Cas9 (CRISPR-SaCas9) has been harnessed as an effective in vivo genome-editing tool to manipulate genomes. However, off-target effects remain a major bottleneck that precludes safe and reliable applications in genome editing. Here, we characterize the off-target effects of wild-type (WT) SaCas9 at single-nucleotide (single-nt) resolution and describe a directional screening system to identify novel SaCas9 variants with desired properties in human cells. Using this system, we identified enhanced-fidelity SaCas9 (efSaCas9) (variant Mut268 harboring the single mutation of N260D), which could effectively distinguish and reject single base-pair mismatches. We demonstrate dramatically reduced off-target effects (approximately 2- to 93-fold improvements) of Mut268 compared to WT using targeted deep-sequencing analyses. To understand the structural origin of the fidelity enhancement, we find that N260, located in the REC3 domain, orchestrates an extensive network of contacts between REC3 and the guide RNA-DNA heteroduplex. efSaCas9 can be broadly used in genome-editing applications that require high fidelity. Furthermore, this study provides a general strategy to rapidly evolve other desired CRISPR-Cas9 traits besides enhanced fidelity, to expand the utility of the CRISPR toolkit.


Asunto(s)
Proteínas Bacterianas/metabolismo , Proteína 9 Asociada a CRISPR/metabolismo , Staphylococcus aureus/metabolismo , Biblioteca de Genes , Ingeniería Genética , Sitios Genéticos , Genoma Humano , Células HEK293 , Humanos , Nucleótidos/genética , Fenotipo , Reproducibilidad de los Resultados , Activación Transcripcional/genética
2.
Hum Mol Genet ; 27(24): 4303-4314, 2018 12 15.
Artículo en Inglés | MEDLINE | ID: mdl-30239724

RESUMEN

Polyglutamine (polyQ) repeat diseases are a class of neurodegenerative disorders caused by CAG-repeat expansion. There are diverse cellular mechanisms behind the pathogenesis of polyQ disorders, including transcriptional dysregulation. Interestingly, we find that levels of the long isoform of nuclear paraspeckle assembly transcript 1 (Neat1L) are elevated in the brains of mouse models of spinocerebellar ataxia types 1, 2, 7 and Huntington's disease (HD). Neat1L was also elevated in differentiated striatal neurons derived from HD knock-in mice and in HD patient brains. The elevation was mutant Huntingtin (mHTT) dependent, as knockdown of mHTT in vitro and in vivo restored Neat1L to normal levels. In additional studies, we found that Neat1L is repressed by methyl CpG binding protein 2 (MeCP2) by RNA-protein interaction but not by occupancy of MeCP2 at its promoter. We also found that NEAT1L overexpression protects from mHTT-induced cytotoxicity, while reducing it enhanced mHTT-dependent toxicity. Gene set enrichment analysis of previously published RNA sequencing data from mouse embryonic fibroblasts and cells derived from HD patients shows that loss of NEAT1L impairs multiple cellular functions, including pathways involved in cell proliferation and development. Intriguingly, the genes dysregulated in HD human brain samples overlap with pathways affected by a reduction in NEAT1, confirming the correlation of NEAT1L and HD-induced perturbations. Cumulatively, the role of NEAT1L in polyQ disease model systems and human tissues suggests that it may play a protective role in CAG-repeat expansion diseases.


Asunto(s)
Enfermedad de Huntington/genética , Proteína 2 de Unión a Metil-CpG/genética , ARN Largo no Codificante/genética , Ataxias Espinocerebelosas/genética , Empalme Alternativo/genética , Animales , Diferenciación Celular/genética , Cuerpo Estriado/metabolismo , Cuerpo Estriado/patología , Técnicas de Sustitución del Gen , Humanos , Proteína Huntingtina/genética , Enfermedad de Huntington/fisiopatología , Ratones , Neuronas/metabolismo , Neuronas/patología , Péptidos/genética , Regiones Promotoras Genéticas , Isoformas de Proteínas/genética , Proteínas de Unión al ARN/genética , Ataxias Espinocerebelosas/fisiopatología , Expansión de Repetición de Trinucleótido/genética
3.
Nucleic Acids Res ; 44(15): 7120-31, 2016 09 06.
Artículo en Inglés | MEDLINE | ID: mdl-27418678

RESUMEN

MicroRNAs (miRs) have emerged as key biological effectors in human health and disease. These small noncoding RNAs are incorporated into Argonaute (Ago) proteins, where they direct post-transcriptional gene silencing via base-pairing with target transcripts. Although miRs have become intriguing biological entities and attractive therapeutic targets, the translational impacts of miR research remain limited by a paucity of empirical miR targeting data, particularly in human primary tissues. Here, to improve our understanding of the diverse roles miRs play in cardiovascular function and disease, we applied high-throughput methods to globally profile miR:target interactions in human heart tissues. We deciphered Ago2:RNA interactions using crosslinking immunoprecipitation coupled with high-throughput sequencing (HITS-CLIP) to generate the first transcriptome-wide map of miR targeting events in human myocardium, detecting 4000 cardiac Ago2 binding sites across >2200 target transcripts. Our initial exploration of this interactome revealed an abundance of miR target sites in gene coding regions, including several sites pointing to new miR-29 functions in regulating cardiomyocyte calcium, growth and metabolism. Also, we uncovered several clinically-relevant interactions involving common genetic variants that alter miR targeting events in cardiomyopathy-associated genes. Overall, these data provide a critical resource for bolstering translational miR research in heart, and likely beyond.


Asunto(s)
Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Reactivos de Enlaces Cruzados , Inmunoprecipitación , MicroARNs/metabolismo , Miocardio/metabolismo , Transcriptoma/genética , Regiones no Traducidas 3'/genética , Sitios de Unión , Calcio/metabolismo , Cardiomiopatías/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Miocardio/citología , Sistemas de Lectura Abierta/genética , Polimorfismo de Nucleótido Simple/genética , Especificidad por Sustrato
5.
HGG Adv ; 4(1): 100146, 2023 01 12.
Artículo en Inglés | MEDLINE | ID: mdl-36262216

RESUMEN

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by CAG trinucleotide repeat expansions in exon-1 of huntingtin (HTT). Currently, there is no cure for HD, and the clinical care of individuals with HD is focused on symptom management. Previously, we showed allele-specific deletion of the expanded HTT allele (mHTT) using CRISPR-Cas9 by targeting nearby (<10 kb) SNPs that created or eliminated a protospacer adjacent motif (PAM) near exon-1. Here, we comprehensively analyzed all potential PAM sites within a 10.4-kb genomic region flanking exon-1 of HTT in 983 individuals with HD using a multiplex targeted long-read sequencing approach on the Oxford Nanopore platform. We developed computational tools (NanoBinner and NanoRepeat) to de-multiplex the data, detect repeats, and phase the reads on the expanded or the wild-type HTT allele. One SNP common to 30% of individuals with HD of European ancestry emerged through this analysis, which was confirmed as a strong candidate for allele-specific deletion of the mHTT in human HD cell lines. In addition, up to 57% HD individuals may be candidates for allele-specific editing through combinatorial SNP targeting. Cumulatively, we provide a haplotype map of the region surrounding exon-1 of HTT in individuals affected with HD. Our workflow can be applied to other repeat expansion diseases to facilitate the design of guide RNAs for allele-specific gene editing.


Asunto(s)
Edición Génica , Enfermedades Neurodegenerativas , Humanos , Alelos , Polimorfismo de Nucleótido Simple/genética , Haplotipos/genética
6.
Nat Commun ; 14(1): 2628, 2023 05 06.
Artículo en Inglés | MEDLINE | ID: mdl-37149717

RESUMEN

Alternative splicing of neuronal genes is controlled partly by the coordinated action of polypyrimidine tract binding proteins (PTBPs). While PTBP1 is ubiquitously expressed, PTBP2 is predominantly neuronal. Here, we define the PTBP2 footprint in the human transcriptome using brain tissue and human induced pluripotent stem cell-derived neurons (iPSC-neurons). We map PTBP2 binding sites, characterize PTBP2-dependent alternative splicing events, and identify novel PTBP2 targets including SYNGAP1, a synaptic gene whose loss-of-function leads to a complex neurodevelopmental disorder. We find that PTBP2 binding to SYNGAP1 mRNA promotes alternative splicing and nonsense-mediated decay, and that antisense oligonucleotides (ASOs) that disrupt PTBP binding redirect splicing and increase SYNGAP1 mRNA and protein expression. In SYNGAP1 haploinsufficient iPSC-neurons generated from two patients, we show that PTBP2-targeting ASOs partially restore SYNGAP1 expression. Our data comprehensively map PTBP2-dependent alternative splicing in human neurons and cerebral cortex, guiding development of novel therapeutic tools to benefit neurodevelopmental disorders.


Asunto(s)
Células Madre Pluripotentes Inducidas , Proteínas del Tejido Nervioso , Humanos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Empalme del ARN , Empalme Alternativo/genética , Encéfalo/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Activadoras de ras GTPasa/genética , Ribonucleoproteínas Nucleares Heterogéneas/genética , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Proteína de Unión al Tracto de Polipirimidina/genética , Proteína de Unión al Tracto de Polipirimidina/metabolismo
7.
Biotechnol J ; 14(7): e1800689, 2019 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-30927491

RESUMEN

Genome editing using RNA-guided nucleases in their ribonucleoprotein (RNP) form represents a promising strategy for gene modification and therapy because they are free of exogenous DNA integration and have reduced toxicity in vivo and ex vivo. However, genome editing by Cas9 nuclease from Staphylococcus aureus (SaCas9) has not been reported in its RNP form, which recognizes a longer protospacer adjacent motif (PAM), 5'-NNGRRT-3', compared with Streptococcus pyogenes Cas9 (SpCas9) of 5'-NGG-3' PAM. Here, SaCas9-RNP-mediated genome editing is reported in human cells. The SaCas9-RNP displayed efficient genome editing activities of enhanced green fluorescent protein (EGFP) coding gene as well as three endogenous genes (OPA1, RS1, and VEGFA). Further, SaCas9-RNP is successfully implemented to correct a pathogenic RS1 mutation for X-linked juvenile retinoschisis. It is also shown that off-target effects triggered by SaCas9-RNP are undetectable by targeted deep sequencing. Collectively, this study demonstrates the potential of SaCas9-RNP-mediated genome editing in human cells, which could facilitate genome-editing-based therapy.


Asunto(s)
Proteínas Bacterianas , Proteína 9 Asociada a CRISPR , Edición Génica/métodos , Genoma Humano/genética , Ribonucleoproteínas , Staphylococcus aureus/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/aislamiento & purificación , Proteína 9 Asociada a CRISPR/metabolismo , Escherichia coli/genética , Células HEK293 , Humanos , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo
8.
Biochem Biophys Res Commun ; 366(3): 670-4, 2008 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-18078808

RESUMEN

New therapeutics are urgently needed for the treatment of pandemic influenza caused by H5N1 influenza virus mutants. Aptamer was a promising candidate for treatment and prophylaxis of influenza virus infections. In this study, systemic evolution of ligands through exponential enrichment (SELEX) was used to screen DNA aptamers targeted to recombinant HA1 proteins of the H5N1 influenza virus. After 11 rounds of selection, DNA aptamers that bind to the HA1 protein were isolated and shown to have different binding capacities. Among them, aptamer 10 had the strongest binding to the HA1 protein, and had an inhibitory effect on H5N1 influenza virus, as shown by the hemagglutinin and MTT assays. These results should aid the development of new drugs for the prevention and control of influenza virus infections.


Asunto(s)
Virus ADN/genética , Subtipo H5N1 del Virus de la Influenza A/fisiología , Gripe Humana/virología , Técnica SELEX de Producción de Aptámeros , Vacunas de ADN/administración & dosificación , Vacunas de ADN/genética , Inactivación de Virus/efectos de los fármacos , Humanos , Subtipo H5N1 del Virus de la Influenza A/efectos de los fármacos , Gripe Humana/tratamiento farmacológico
9.
Wei Sheng Wu Xue Bao ; 47(3): 418-22, 2007 Jun.
Artículo en Zh | MEDLINE | ID: mdl-17672298

RESUMEN

NS1 gene was amplified from an H5N1 influenza virus, A/Anhui/01/2005 for cloning, sequence analysis and later expression in Escherichia coli. The result showed that NS1 of A/Anhui/01/2005 strain had the close phylogenetic relationship with that of H5N1 avian influenza strains recently isolated in Fujian and Hunan. Correspondingly, its amino acid sequence showed the highest homology with that of Fujian and Hunan strains. The amino acid position of 92 involved in virus virulence was Asp, contrast to Glu in A/HK/156/97. Five amino acid deletion from 80 to 84 was also found in A/Anhui/01/2005, which was considered as a contributor to virus resistance against cytokine, such as IFN, TNF, etc. A motif binding to CBSF, conveted to GFWEN, which was different from previous GLEWN found in other 19 strains. Besides, the PL motif of ESEV, binding to PDZ domain, is the same as previous high-mortality H5N1 isolates. Furthermore, this NS1 was efficiently expressed in Escherichia coli and the highly purified product demonstrated wonderful activity as confirmed by Western blot. As a result, the work paves the way for further understanding the role of NSI in human H5N1 infection and development of new antiviral drugs against influenza virus.


Asunto(s)
Escherichia coli/genética , Expresión Génica , Subtipo H5N1 del Virus de la Influenza A/genética , Subtipo H5N1 del Virus de la Influenza A/aislamiento & purificación , Gripe Humana/virología , Proteínas no Estructurales Virales/genética , China , Escherichia coli/metabolismo , Humanos , Subtipo H5N1 del Virus de la Influenza A/química , Subtipo H5N1 del Virus de la Influenza A/clasificación , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Filogenia , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/metabolismo
10.
Wei Sheng Wu Xue Bao ; 45(2): 292-7, 2005 Apr.
Artículo en Zh | MEDLINE | ID: mdl-15989279

RESUMEN

The biosafety of DNA vaccine is one of the key questions which should be solved before it is used in the clinical trail. In order to evaluate the biosafety of DNA vaccine, the CSFV DNA vaccine was used in the studying target, two main aspects of the vaccine were explored in the study. Firstly, the possibility of integration of two kinds of DNA vaccine plasmids into pig genome was analyzed by PCR technology after the different vaccines were injected through the intramuscular introduction. The results showed that both plasmids DNA were detected as the form were not integrated into pig genome, it can be detected 30 copies plasmid DNA in 1 microg total genomic DNA as the sensibility of PCR, indicated the safety of the DNA vaccine. Afterward the environmental fecal and soil samples in the experimental pens were picked up. Then the antibiotic resistant bacteria were isolated and its resistant genes were analyzed by PCR and gene sequencing. The results demonstrated that the transfer and spreading of two DNA vaccine plasmids studied into environmental bacteria from receptor pigs were not found. The results showed that the CSFV DNA vaccine is safe to both pigs and the surrounding environment.


Asunto(s)
Virus de la Fiebre Porcina Clásica/inmunología , Vacunas de ADN/efectos adversos , Vacunas Virales/efectos adversos , Animales , Virus de la Fiebre Porcina Clásica/genética , Genoma Viral , Plásmidos/genética , Reacción en Cadena de la Polimerasa , Seguridad , Porcinos , Vacunas de ADN/genética , Vacunas Virales/genética
11.
Mol Ther Nucleic Acids ; 2: e67, 2013 Jan 08.
Artículo en Inglés | MEDLINE | ID: mdl-23299833

RESUMEN

The physiological barriers of the brain impair drug delivery for treatment of many neurological disorders. One delivery approach that has not been investigated for their ability to penetrate the brain is RNA-based aptamers. These molecules can impart delivery to peripheral tissues and circulating immune cells, where they act as ligand mimics or can be modified to carry payloads. We developed a library of aptamers and an in vivo evolution protocol to determine whether specific aptamers could be identified that would home to the brain after injection into the peripheral vasculature. Unlike biopanning with recombinant bacteriophage libraries, we found that the aptamer library employed here required more than 15 rounds of in vivo selection for convergence to specific sequences. The aptamer species identified through this approach bound to brain capillary endothelia and penetrated into the parenchyma. The methods described may find general utility for targeting various payloads to the brain.Molecular Therapy - Nucleic Acids (2013) 2, e67; doi:10.1038/mtna.2012.59; published online 8 January 2013.

12.
Artículo en Zh | MEDLINE | ID: mdl-22106475

RESUMEN

OBJECTIVE: To generate the Escherichia col vector expressing human H5N1 influenza virus M1 protein. To provide useful tools for detection of human H5N1 influenza virus and study on biological function of M1 protein. METHODS: M1 gene fragment was amplified by PCR using the influenza virus gene segment 7 as template, and was subcloned into pQE80-L vector. The recombinant plasmid pQE80-L/M1 was transformed into Escherichia coil BL21 (DE3) strain. The expression of M1 was induced by isopropy-beta3-D-thiogalactopyranoside. We purified the recombinant M1 protein with polyhistidine tag with Ni2+ affinity chromatography. Mouse were immunized with the purified M1 protein for preparing antibodies against M1. RESULTS: The recombinant Ml protein was recognized by antiserum against H5N1 subtype influenza virus, elicit specific antibody in immunized animals. CONCLUSION: These confirmed that we successfully constructed the Escherichia coli vector expressing human H5N1 influenza virus M1 protein.


Asunto(s)
Clonación Molecular , Escherichia coli/genética , Expresión Génica , Subtipo H5N1 del Virus de la Influenza A/genética , Proteínas de la Matriz Viral/genética , Animales , Escherichia coli/metabolismo , Humanos , Inmunización , Subtipo H5N1 del Virus de la Influenza A/inmunología , Gripe Humana/inmunología , Ratones , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas de la Matriz Viral/inmunología
13.
J Drug Target ; 17(2): 133-9, 2009 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-19089690

RESUMEN

Influenza, mainly caused by influenza virus, is becoming one of the major concerns in the world. Limitation in vaccines necessitates the urgent development of new therapeutic options against this virus. In the present study, we designed small interfering RNA (siRNA) targeting overlapping gene of PB1 and PB1-F2 gene of the influenza A virus and investigated its effect against influenza A virus infection. A reduction in virus-associated cell apoptosis was observed in A549 cells treated with this siRNA. Furthermore, its antiviral effect was confirmed by different methods. Also, a marked decrease of virus titer in chicken embryos treated with the siRNA was observed. The findings of this work highlight the potential of this shared region to be an additional therapeutic target for the treatment of influenza virus infection.


Asunto(s)
Subtipo H1N1 del Virus de la Influenza A/fisiología , ARN Interferente Pequeño/genética , Proteínas Virales/genética , Animales , Apoptosis , Línea Celular , Embrión de Pollo , Perros , Genoma Viral , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H5N1 del Virus de la Influenza A/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección , Proteínas Virales/biosíntesis , Replicación Viral
14.
Bing Du Xue Bao ; 23(5): 366-70, 2007 Sep.
Artículo en Zh | MEDLINE | ID: mdl-17969853

RESUMEN

Based on the first isolated human H5N1 influenza virus strain A/Anhui/1/2005 in China, HA and HA1 genes were amplified and cloned into the eukaryotic expression vector pStar. The recombinant plasmids pStar-HA and pStar-HA1 were transfected into COS7 cells. Western blot and IFA showed that the two recombinant DNA plasmids were successfully expressed in eukaryotic cells. BALB/c mice were immunized with the plasmids DNA by intramuscular injection. Anti-HA specific antibody in peripheral blood of immunized mice was tested by ELISA. The results showed that the recombinant plasmids successfully induced the anti-HA humoral immune response, and there was no significant difference between HA and HA1 as immunogen. This work provides basis for future development of novel avian flu vaccine.


Asunto(s)
Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Subtipo H5N1 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Vacunas de ADN/inmunología , Animales , Anticuerpos Antivirales/sangre , Células COS , Chlorocebus aethiops , Femenino , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Inmunoglobulina G/sangre , Ratones , Ratones Endogámicos BALB C
15.
Cytokine ; 36(5-6): 296-304, 2006 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-17379534

RESUMEN

Tumor necrosis factor alpha plays primary role in the pathogenesis of inflammatory diseases. TNFalpha is essential for antigen-specific IgE production and for the induction of Th2-type cytokines. The lack of TNFalpha inhibited the development of allergic rhinitis. In this study, the chimeric gene of soluble TNF receptor and IgGFc fragment (sTNFR-IgGFc) was cloned into the EBV-based plasmid pGEG. When the plasmid pGEG.sTNFR-IgGFc was transferred to endothelium cell, a considerable expression of the sTNFR-IgGFc fusion protein was detected. Moreover, the expression product in the supernatant could antagonize the cytolytic activity of TNFalpha on L929 cells. Then the plasmid was delivered into nasal mucosa of allergic rhinitis mice to determine its effect on this animal model. Results showed that symptoms in treated group were improved. Pathological examination showed the numbers of eosinophil, mast cell and IL-5(+) cells in treated groups were reduced compared with placebo group. These data showed that pGEG.sTNFR-IgGFc expression plasmid is potential for the treatment of allergic rhinitis, and suggest that the antagonist of TNFalpha may provide a new approach for the treatment of allergic rhinitis.


Asunto(s)
Terapia Genética , Fragmentos Fc de Inmunoglobulinas/genética , Inmunoglobulina G/genética , Receptores del Factor de Necrosis Tumoral/genética , Hipersensibilidad Respiratoria/terapia , Rinitis/terapia , Animales , Modelos Animales de Enfermedad , Herpesvirus Humano 4/genética , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Mucosa Nasal/patología , Proteínas Recombinantes/genética , Hipersensibilidad Respiratoria/patología , Rinitis/patología , Factor de Necrosis Tumoral alfa/metabolismo
16.
Sheng Wu Gong Cheng Xue Bao ; 22(3): 378-83, 2006 May.
Artículo en Zh | MEDLINE | ID: mdl-16755914

RESUMEN

Tumor necrosis factor alpha (TNFalpha) is a pro-inflammatory cytokine, acting as a regulator of inflammation and immunity. TNFalpha plays a critical role in the pathogenesis of rheumatoid arthritis. Blocking of TNFa activity suppressed inflammatory tissue damage. In present study, the chimeric gene of soluble TNF receptor and IgG Fc fragment (sTNFR-IgG FC) was cloned into the mammalian cell expression vector pStar. When the plamid pStar/sTNFR-IgGFc-GFP was transfected into endothelial cells, a considerable expression of the sTNFR-IgG Fc fusion protein was detected. Moreover, the product in 100microL expression supernatant could completely antagonize the cytolytic effect of 1ng TNFa on L929 cells, even at 1/64 dilution. Then the plasmid was delivered into CIA-induced rheumatoid arthritis mice by tail vein injection. The expression of sTNFR-IgG Fc was detected in liver by RT-PCR. Animals in treatment group showed reduced symptoms of arthritis and more active. This treatment induced decrease of synovial incrassation and prevented the cartilage destruction of the mice RA model. These results show that tail vein injection is an effective way for gene therapy and sTNFR-IgGFc expression plasmid is potential for the treatment of rheumatoid arthritis.


Asunto(s)
Artritis Reumatoide/terapia , Células Endoteliales/metabolismo , Terapia Genética , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/uso terapéutico , Receptores del Factor de Necrosis Tumoral/biosíntesis , Receptores del Factor de Necrosis Tumoral/uso terapéutico , Animales , Artritis Reumatoide/inducido químicamente , Colágeno Tipo II , Escherichia coli/genética , Escherichia coli/metabolismo , Etanercept , Humanos , Inmunoglobulina G/genética , Masculino , Ratones , Ratones Endogámicos DBA , Receptores del Factor de Necrosis Tumoral/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/uso terapéutico , Transfección , Factor de Necrosis Tumoral alfa/metabolismo
17.
Artículo en Zh | MEDLINE | ID: mdl-16027793

RESUMEN

OBJECTIVE: To explore the role of BM2 protein in the life cycle of influenza B virus. METHODS: The authors screened human kidney MATCHMAKER cDNA library for new binding partners of BM2 of influenza B virus by using the yeast two hybrid system with truncated BM2 (26-109 aa) as the bait. RESULTS: Six positive plasmids encoding N-acetylneuraminate pyruvate lyase, angiopoietin 3, zinc finger protein 251, ribosomal protein S20, protein arginine N-methyltransferase 1 variant 1 (PRMT) and transcription factor-like 1 (TCFL1) were obtained. CONCLUSION: The results suggest that BM2 may play an important role in the life cycle of influenza B virus.


Asunto(s)
Virus de la Influenza B/metabolismo , Técnicas del Sistema de Dos Híbridos , Proteínas Virales/metabolismo , Proteína 1 Similar a la Angiopoyetina , Proteínas Similares a la Angiopoyetina , Angiopoyetinas/genética , Angiopoyetinas/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Biblioteca de Genes , Humanos , Virus de la Influenza B/genética , Riñón/metabolismo , Oxo-Ácido-Liasas/genética , Oxo-Ácido-Liasas/metabolismo , Plásmidos/genética , Unión Proteica , Proteína-Arginina N-Metiltransferasas/genética , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Virales/genética , Dedos de Zinc/genética
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