Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 18.988
Filtrar
1.
Nat Commun ; 11(1): 4903, 2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-32994412

RESUMO

The CRISPR-Cas9 system has increased the speed and precision of genetic editing in cells and animals. However, model generation for drug development is still expensive and time-consuming, demanding more target flexibility and faster turnaround times with high reproducibility. The generation of a tightly controlled ObLiGaRe doxycycline inducible SpCas9 (ODInCas9) transgene and its use in targeted ObLiGaRe results in functional integration into both human and mouse cells culminating in the generation of the ODInCas9 mouse. Genomic editing can be performed in cells of various tissue origins without any detectable gene editing in the absence of doxycycline. Somatic in vivo editing can model non-small cell lung cancer (NSCLC) adenocarcinomas, enabling treatment studies to validate the efficacy of candidate drugs. The ODInCas9 mouse allows robust and tunable genome editing granting flexibility, speed and uniformity at less cost, leading to high throughput and practical preclinical in vivo therapeutic testing.


Assuntos
Sistemas CRISPR-Cas/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Descoberta de Drogas/métodos , Edição de Genes/métodos , Neoplasias Pulmonares/tratamento farmacológico , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Proteína 9 Associada à CRISPR/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Doxiciclina/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Feminino , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Vetores Genéticos/genética , Células HEK293 , Ensaios de Triagem em Larga Escala/métodos , Humanos , Neoplasias Pulmonares/genética , Masculino , Camundongos , Camundongos Transgênicos , RNA Guia/genética , Recombinação Genética/efeitos dos fármacos , Reprodutibilidade dos Testes , Ativação Transcricional/efeitos dos fármacos , Transfecção/métodos , Transgenes/genética
2.
Nat Commun ; 11(1): 4524, 2020 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-32913280

RESUMO

Traumatic brain injury (TBI) is a leading global cause of death and disability. Here we demonstrate in an experimental mouse model of TBI that mild forms of brain trauma cause severe deficits in meningeal lymphatic drainage that begin within hours and last out to at least one month post-injury. To investigate a mechanism underlying impaired lymphatic function in TBI, we examined how increased intracranial pressure (ICP) influences the meningeal lymphatics. We demonstrate that increased ICP can contribute to meningeal lymphatic dysfunction. Moreover, we show that pre-existing lymphatic dysfunction before TBI leads to increased neuroinflammation and negative cognitive outcomes. Finally, we report that rejuvenation of meningeal lymphatic drainage function in aged mice can ameliorate TBI-induced gliosis. These findings provide insights into both the causes and consequences of meningeal lymphatic dysfunction in TBI and suggest that therapeutics targeting the meningeal lymphatic system may offer strategies to treat TBI.


Assuntos
Lesões Encefálicas/fisiopatologia , Gliose/fisiopatologia , Sistema Glinfático/fisiologia , Meninges/fisiopatologia , Animais , Lesões Encefálicas/complicações , Lesões Encefálicas/patologia , Lesões Encefálicas/terapia , Dependovirus/genética , Modelos Animais de Doenças , Feminino , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Gliose/etiologia , Gliose/patologia , Gliose/prevenção & controle , Sistema Glinfático/patologia , Humanos , Masculino , Meninges/patologia , Camundongos , Fator C de Crescimento do Endotélio Vascular/genética , Fator C de Crescimento do Endotélio Vascular/uso terapêutico
3.
Med Microbiol Immunol ; 209(6): 681-691, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32918599

RESUMO

Chimeric Antigen Receptor (CAR)-redirected T cells show great efficacy in the patient-specific therapy of hematologic malignancies. Here, we demonstrate that a DARPin with specificity for CD4 specifically redirects and triggers the activation of CAR engineered T cells resulting in the depletion of CD4+ target cells aiming for elimination of the human immunodeficiency virus (HIV) reservoir.


Assuntos
Repetição de Anquirina , Linfócitos T CD4-Positivos/virologia , Infecções por HIV/imunologia , HIV/isolamento & purificação , Imunoterapia Adotiva , Depleção Linfocítica/métodos , Peptídeos/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Relação Dose-Resposta Imunológica , Avaliação Pré-Clínica de Medicamentos , Gammaretrovirus/genética , Vetores Genéticos/genética , Células HEK293 , Infecções por HIV/virologia , Humanos , Ativação Linfocitária , Peptídeos/química , Anticorpos de Cadeia Única/imunologia , Transdução Genética
5.
Sheng Wu Gong Cheng Xue Bao ; 36(7): 1395-1404, 2020 Jul 25.
Artigo em Chinês | MEDLINE | ID: mdl-32748597

RESUMO

By inserting microRNAs into the intron of EF1α promoter, we constructed a novel lentiviral vector knocking down PD-1 gene via microRNA and applied it to CAR-T cells. Lentiviral transduction efficiency and PD-1-silencing efficiency were detected by flow cytometry. PD-1 expression was detected by Western blotting. Relative expression of microRNA was measured by Q-PCR. Cytotoxicity of CAR-T cells based on this vector was tested by luciferase bioluminescence and flow cytometry. Compared with lentiviral vector with microRNA transcribed by U6 promotor, the transduction efficiency of lentiviral vector with microRNA which was inserted into the intron of EF1α promoter was more significant, and the knockdown rate of PD-1 was more than 90%, which was validated by flow cytometry and Western blotting. And the relative expression level of microRNA in Jurkat cells transduced with this novel lentiviral vector was shown by Q-PCR. Compared with normal CAR-T cells, CAR-T cells based on this vector showed stronger cytotoxicity against PD-L1 positive Raji cells. We successfully constructed a novel lentiviral vector that knocked down PD-1 via microRNA and verified the superiority of its transduction efficiency and knockdown efficiency of PD-1. CAR-T cells based on this vector can exert a more powerful cytotoxicity, thus providing theoretical support for the subsequent treatment of PD-L1 positive tumors.


Assuntos
Vetores Genéticos , Lentivirus , MicroRNAs , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Vetores Genéticos/genética , Humanos , Lentivirus/genética , MicroRNAs/metabolismo , Receptor de Morte Celular Programada 1 , Regiões Promotoras Genéticas/genética
6.
PLoS Pathog ; 16(8): e1008646, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32776974

RESUMO

Inositol hexakisphosphate (IP6) potently stimulates HIV-1 particle assembly in vitro and infectious particle production in vivo. However, knockout cells lacking inositol-pentakisphosphate 2-kinase (IPPK-KO), the enzyme that produces IP6 by phosphorylation of inositol pentakisphosphate (IP5), were still able to produce infectious HIV-1 particles at a greatly reduced rate. HIV-1 in vitro assembly can also be stimulated to a lesser extent with IP5, but until recently, it was not known if IP5 could also function in promoting assembly in vivo. Here we addressed whether there is an absolute requirement for IP6 or IP5 in the production of infectious HIV-1 particles. IPPK-KO cells expressed no detectable IP6 but elevated IP5 levels and displayed a 20-100-fold reduction in infectious particle production, correlating with lost virus release. Transient transfection of an IPPK expression vector stimulated infectious particle production and release in IPPK-KO but not wildtype cells. Several attempts to make IP6/IP5 deficient stable cells were not successful, but transient expression of the enzyme multiple inositol polyphosphate phosphatase-1 (MINPP1) into IPPK-KOs resulted in near ablation of IP6 and IP5. Under these conditions, we found that HIV-1 infectious particle production and virus release were essentially abolished (1000-fold reduction) demonstrating an IP6/IP5 requirement. However, other retroviruses including a Gammaretrovirus, a Betaretrovirus, and two non-primate Lentiviruses displayed only a modest (3-fold) reduction in infectious particle production from IPPK-KOs and were not significantly altered by expression of IPPK or MINPP1. The only other retrovirus found to show a clear IP6/IP5 dependence was the primate (macaque) Lentivirus Simian Immunodeficiency Virus, which displayed similar sensitivity as HIV-1. We were not able to determine if producer cell IP6/IP5 is required at additional steps beyond assembly because viral particles devoid of both molecules could not be generated. Finally, we found that loss of IP6/IP5 in viral target cells had no effect on permissivity to HIV-1 infection.


Assuntos
Vetores Genéticos/administração & dosagem , Infecções por HIV/virologia , Fosfatos de Inositol/metabolismo , Lentivirus de Primatas/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Ácido Fítico/metabolismo , Vírion/fisiologia , Animais , Vetores Genéticos/genética , HIV/fisiologia , Infecções por HIV/metabolismo , Infecções por HIV/patologia , Humanos , Fosforilação , Primatas
7.
PLoS Pathog ; 16(8): e1008326, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32804988

RESUMO

CRISPR/Cas has become the state-of-the-art technology for genetic manipulation in diverse organisms, enabling targeted genetic changes to be performed with unprecedented efficiency. Here we report on the first establishment of robust CRISPR/Cas editing in the important necrotrophic plant pathogen Botrytis cinerea based on the introduction of optimized Cas9-sgRNA ribonucleoprotein complexes (RNPs) into protoplasts. Editing yields were further improved by development of a novel strategy that combines RNP delivery with cotransformation of transiently stable vectors containing telomeres, which allowed temporary selection and convenient screening for marker-free editing events. We demonstrate that this approach provides superior editing rates compared to existing CRISPR/Cas-based methods in filamentous fungi, including the model plant pathogen Magnaporthe oryzae. Genome sequencing of edited strains revealed very few additional mutations and no evidence for RNP-mediated off-targeting. The high performance of telomere vector-mediated editing was demonstrated by random mutagenesis of codon 272 of the sdhB gene, a major determinant of resistance to succinate dehydrogenase inhibitor (SDHI) fungicides by in bulk replacement of the codon 272 with codons encoding all 20 amino acids. All exchanges were found at similar frequencies in the absence of selection but SDHI selection allowed the identification of novel amino acid substitutions which conferred differential resistance levels towards different SDHI fungicides. The increased efficiency and easy handling of RNP-based cotransformation is expected to accelerate molecular research in B. cinerea and other fungi.


Assuntos
Botrytis/fisiologia , Sistemas CRISPR-Cas , Edição de Genes , Oryza/microbiologia , Doenças das Plantas/microbiologia , Ribonucleoproteínas/antagonistas & inibidores , Telômero/genética , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Oryza/genética , Doenças das Plantas/genética , Ribonucleoproteínas/genética
8.
PLoS One ; 15(8): e0236477, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32756607

RESUMO

Antibodies function by binding to antigens. Antibodies must be cloned and expressed to determine their binding characteristics, but current methods for high-throughput antibody sequencing yield antibody DNA pooled from many cells and do not readily permit cloning of antibodies from single B cells. We present a strategy for retrieving and cloning antibody DNA from single cells within a pooled library of cells. Our strategy, called selective PCR for antibody retrieval (SPAR), takes advantage of the unique sequence barcodes attached to individual cDNA molecules during sample preparation to enable specific amplification by PCR of antibody heavy- and light-chain cDNA originating from a single cell. We show through computational analysis that most human antibodies sequenced using typical high-throughput methods can be retrieved using SPAR, and experimentally demonstrate retrieval of full-length antibody variable region cDNA from three cells within pools of ~5,000 cells. SPAR enables rapid low-cost cloning and expression of native human antibodies from pooled single-cell sequence libraries for functional characterization.


Assuntos
Anticorpos/genética , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Reação em Cadeia da Polimerase/métodos , Sequência de Aminoácidos/genética , Técnicas de Visualização da Superfície Celular , Clonagem Molecular , DNA Complementar/genética , Biblioteca Gênica , Vetores Genéticos/genética , Humanos , Análise de Célula Única
9.
Int J Mol Sci ; 21(14)2020 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-32698494

RESUMO

Single-stranded RNA viruses such as alphaviruses, flaviviruses, measles viruses and rhabdoviruses are characterized by their capacity of highly efficient self-amplification of RNA in host cells, which make them attractive vehicles for vaccine development. Particularly, alphaviruses and flaviviruses can be administered as recombinant particles, layered DNA/RNA plasmid vectors carrying the RNA replicon and even RNA replicon molecules. Self-amplifying RNA viral vectors have been used for high level expression of viral and tumor antigens, which in immunization studies have elicited strong cellular and humoral immune responses in animal models. Vaccination has provided protection against challenges with lethal doses of viral pathogens and tumor cells. Moreover, clinical trials have demonstrated safe application of RNA viral vectors and even promising results in rhabdovirus-based phase III trials on an Ebola virus vaccine. Preclinical and clinical applications of self-amplifying RNA viral vectors have proven efficient for vaccine development and due to the presence of RNA replicons, amplification of RNA in host cells will generate superior immune responses with significantly reduced amounts of RNA delivered. The need for novel and efficient vaccines has become even more evident due to the global COVID-19 pandemic, which has further highlighted the urgency in challenging emerging diseases.


Assuntos
Neoplasias/prevenção & controle , RNA Viral/genética , Vacinas Sintéticas/imunologia , Vacinas Virais/imunologia , Viroses/prevenção & controle , Animais , Vetores Genéticos/genética , Humanos , Camundongos , Neoplasias/virologia , Vacinação , Vírus/genética
10.
Nat Commun ; 11(1): 3279, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32606306

RESUMO

Recombinant adeno-associated viruses (rAAVs) are currently considered the safest and most reliable gene delivery vehicles for human gene therapy. Three serotype capsids, AAV1, AAV2, and AAV9, have been approved for commercial use in patients, but they may not be suitable for all therapeutic contexts. Here, we describe a novel capsid identified in a human clinical sample by high-throughput, long-read sequencing. The capsid, which we have named AAVv66, shares high sequence similarity with AAV2. We demonstrate that compared to AAV2, AAVv66 exhibits enhanced production yields, virion stability, and CNS transduction. Unique structural properties of AAVv66 visualized by cryo-EM at 2.5-Å resolution, suggest that critical residues at the three-fold protrusion and at the interface of the five-fold axis of symmetry likely contribute to the beneficial characteristics of AAVv66. Our findings underscore the potential of AAVv66 as a gene therapy vector.


Assuntos
Proteínas do Capsídeo/genética , Capsídeo/metabolismo , Dependovirus/genética , Vetores Genéticos/genética , Animais , Capsídeo/ultraestrutura , Proteínas do Capsídeo/classificação , Sistema Nervoso Central/virologia , Microscopia Crioeletrônica , DNA Viral/análise , DNA Viral/genética , Dependovirus/classificação , Dependovirus/fisiologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Filogenia , Sorogrupo , Transdução Genética , Montagem de Vírus/genética
11.
Nat Biotechnol ; 38(7): 845-855, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32601435

RESUMO

Genome editing has the potential to treat an extensive range of incurable monogenic and complex diseases. In particular, advances in sequence-specific nuclease technologies have dramatically accelerated the development of therapeutic genome editing strategies that are based on either the knockout of disease-causing genes or the repair of endogenous mutated genes. These technologies are progressing into human clinical trials. However, challenges remain before the therapeutic potential of genome editing can be fully realized. Delivery technologies that have serendipitously been developed over the past couple decades in the protein and nucleic acid delivery fields have been crucial to genome editing success to date, including adeno-associated viral and lentiviral vectors for gene therapy and lipid nanoparticle and other non-viral vectors for nucleic acid and protein delivery. However, the efficiency and tissue targeting capabilities of these vehicles must be further improved. In addition, the genome editing enzymes themselves need to be optimized, and challenges regarding their editing efficiency, specificity and immunogenicity must be addressed. Emerging protein engineering and synthetic chemistry approaches can offer solutions and enable the development of safe and efficacious clinical genome editing.


Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes/tendências , Doenças Genéticas Inatas/terapia , Terapia Genética , Doenças Genéticas Inatas/genética , Vetores Genéticos/genética , Vetores Genéticos/uso terapêutico , Humanos , Nanopartículas/uso terapêutico , Engenharia de Proteínas
12.
PLoS Negl Trop Dis ; 14(7): e0008459, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32667913

RESUMO

Rabies, caused by RNA viruses in the Genus Lyssavirus, is the most fatal of all infectious diseases. This neglected zoonosis remains a major public health problem in developing countries, causing the death of an estimated 25,000-159,000 people each year, with more than half of them in children. The high incidence of human rabies in spite of effective vaccines is mainly linked to the lack of compliance with the complicated administration schedule, inadequacies of the community public health system for local administration by the parenteral route and the overall costs of the vaccine. The goal of our work was the development of a simple, affordable and effective vaccine strategy to prevent human rabies virus infection. This next generation vaccine is based on a replication-defective chimpanzee adenovirus vector belonging to group C, ChAd155-RG, which encodes the rabies glycoprotein (G). We demonstrate here that a single dose of this vaccine induces protective efficacy in a murine model of rabies challenge and elicits strong and durable neutralizing antibody responses in vaccinated non-human primates. Importantly, we demonstrate that one dose of a commercial rabies vaccine effectively boosts the neutralizing antibody responses induced by ChAd155-RG in vaccinated monkeys, showing the compatibility of the novel vectored vaccine with the current post-exposure prophylaxis in the event of rabies virus exposure. Finally, we demonstrate that antibodies induced by ChAd155-RG can also neutralize European bat lyssaviruses 1 and 2 (EBLV-1 and EBLV-2) found in bat reservoirs.


Assuntos
Adenovirus dos Símios/genética , Vacinas Antirrábicas/imunologia , Raiva/prevenção & controle , Animais , Antígenos Virais , Feminino , Vetores Genéticos/genética , Humanos , Macaca fascicularis , Camundongos , Pan troglodytes/virologia , Profilaxia Pós-Exposição , Coelhos , Vírus da Raiva/genética , Vírus da Raiva/imunologia , Sorogrupo , Vacinação , Vacinas Sintéticas/imunologia , Zoonoses
13.
PLoS One ; 15(6): e0235362, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32584895

RESUMO

OBJECTIVE: Cardiovascular disease is a leading cause of death worldwide. Obesity-related metabolic disorders including dyslipidemia cause impaired collateralization under ischemic conditions, thereby resulting in exacerbated cardiovascular dysfunction. Pemafibrate is a novel selective PPARα modulator, which has been reported to improve atherogenic dyslipidemia, in particular, hypertriglyceridemia and low HDL-cholesterol. Here, we investigated whether pemafibrate modulates the revascularization process in a mouse model of hindlimb ischemia. METHODS AND RESULTS: Male wild-type (WT) mice were randomly assigned to two groups, normal diet or pemafibrate admixture diet from the ages of 6 weeks. After 4 weeks, mice were subjected to unilateral hindlimb surgery to remove the left femoral artery and vein. Pemafibrate treatment enhanced blood flow recovery and capillary formation in ischemic limbs of mice, which was accompanied by enhanced phosphorylation of endothelial nitric oxide synthase (eNOS). Treatment of cultured endothelial cells with pemafibrate resulted in increased network formation and migratory activity, which were blocked by pretreatment with the NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME). Pemafibrate treatment also increased plasma levels of the PPARα-regulated gene, fibroblast growth factor (FGF) 21 in WT mice. Systemic administration of adenoviral vectors expressing FGF21 (Ad-FGF21) to WT mice enhanced blood flow recovery, capillary density and eNOS phosphorylation in ischemic limbs. Treatment of cultured endothelial cells with FGF21 protein led to increases in endothelial cell network formation and migration, which were canceled by pretreatment with L-NAME. Furthermore, administration of pemafibrate or Ad-FGF21 had no effects on blood flow in ischemic limbs in eNOS-deficient mice. CONCLUSION: These data suggest that pemafibrate can promote revascularization in response to ischemia, at least in part, through direct and FGF21-mediated modulation of endothelial cell function. Thus, pemafibrate could be a potentially beneficial drug for ischemic vascular disease.


Assuntos
Benzoxazóis/farmacologia , Butiratos/farmacologia , Isquemia/patologia , Neovascularização Fisiológica/efeitos dos fármacos , Óxido Nítrico Sintase Tipo III/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Fatores de Crescimento de Fibroblastos/sangue , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Membro Posterior/irrigação sanguínea , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Camundongos , Camundongos Knockout , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , PPAR alfa/química , PPAR alfa/metabolismo , Fosforilação/efeitos dos fármacos
14.
PLoS Pathog ; 16(6): e1008522, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32589686

RESUMO

DNA vectors have been widely used as a priming of poxvirus vaccine in prime/boost regimens. Whether the number of DNA impacts qualitatively or quantitatively the immune response is not fully explored. With the aim to reinforce T-cell responses by optimizing the prime-boost regimen, the multicentric EV03/ANRS VAC20 phase I/II trial, randomized 147 HIV-negative volunteers to either 3xDNA plus 1xNYVAC (weeks 0, 4, 8 plus 24; n = 74) or to 2xDNA plus 2xNYVAC (weeks 0, 4 plus 20, 24; n = 73) groups. T-cell responses (IFN-γ ELISPOT) to at least one peptide pool were higher in the 3xDNA than the 2xDNA groups (91% and 80% of vaccinees) (P = 0.049). In the 3xDNA arm, 26 (37%) recipients developed a broader T-cell response (Env plus at least to one of the Gag, Pol, Nef pools) than in the 2xDNA (15; 22%) arms (primary endpoint; P = 0.047) with a higher magnitude against Env (at week 26) (P<0.001). In both groups, vaccine regimens induced HIV-specific polyfunctional CD4 and CD8 T cells and the production of Th1, Th2 and Th17/IL-21 cytokines. Antibody responses were also elicited in up to 81% of vaccines. A higher percentage of IgG responders was noted in the 2xDNA arm compared to the 3xDNA arm, while the 3xDNA group tended to elicit a higher magnitude of IgG3 response against specific Env antigens. We show here that the modulation of the prime strategy, without modifying the route or the dose of administration, or the combination of vectors, may influence the quality of the responses.


Assuntos
Vacinas contra a AIDS/imunologia , Vetores Genéticos/imunologia , Antígenos HIV/imunologia , Poxviridae/imunologia , Vacinas de DNA/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Vacinas contra a AIDS/administração & dosagem , Vacinas contra a AIDS/genética , Adolescente , Adulto , Linfócitos T CD8-Positivos/imunologia , Feminino , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Antígenos HIV/administração & dosagem , Antígenos HIV/genética , Humanos , Interferon gama/imunologia , Masculino , Pessoa de Meia-Idade , Poxviridae/genética , Linfócitos T Auxiliares-Indutores/metabolismo , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genética , Produtos do Gene env do Vírus da Imunodeficiência Humana/administração & dosagem , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética
15.
Sheng Wu Gong Cheng Xue Bao ; 36(6): 1232-1240, 2020 Jun 25.
Artigo em Chinês | MEDLINE | ID: mdl-32597073

RESUMO

Overlap extension PCR is a common method for site-directed mutagenesis. As objective gene sequence growing longer, it is often difficult to obtain the target product in the second round of PCR, and it is highly possible to introduce unexpected mutations into a long gene fragment by PCR. To circumvent these problems, we can only amplify a small gene fragment which contain the target mutation by overlap extension PCR, and then ligate it with vector to get target plasmid. If the restriction site at the end of the amplified fragment was not a single one on plasmid vector, double fragments ligation method could be used to construct target plasmid. Partial amplification, combined with double fragments ligation, could solve lots of problems in long gene mutagenesis. Taking retinoblastoma gene 1 S780E mutagenesis as an example, it is difficult to amplify whole retinoblastoma gene 1 by overlap extension PCR because of long fragment interfering the overlapping extension of second round PCR. However, it is relatively easy to amplify the F3 (1 968-2 787) fragment which contains target mutation S780E. There is a Nhe I site which can be used for ligation on 5' end of F3 fragment, but another Nhe I site on the plasmid restrained from doing so directly. In order to circumvent this obstacle, we ligated F3 fragment, combining with F2 (900-1 968) fragment which was digested from wild type plasmid, with the vector which contain F1 (1-900) fragment of the gene. That double fragments ligated with one vector at the same time, though less efficient, can recombine into a complete plasmid. The sequences of the two selected recombinant plasmids were consistent with the target mutation, which verified the feasibility of this scheme. As an improvement of overlap extension PCR, partial amplification and double fragments ligation methods could provide solutions for site directed mutagenesis of many long genes.


Assuntos
Vetores Genéticos , Mutagênese Sítio-Dirigida , Técnicas de Amplificação de Ácido Nucleico , Sequência de Bases , Clonagem Molecular , Vetores Genéticos/genética , Mutagênese Sítio-Dirigida/métodos , Plasmídeos , Reação em Cadeia da Polimerase
16.
J Vis Exp ; (159)2020 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-32478714

RESUMO

Transgenic animal models are fundamentally important for modern biomedical research. The incorporation of foreign genes into early mouse or rat embryos is an invaluable tool for gene function analysis in living organisms. The standard transgenesis method is based on microinjecting foreign DNA fragments into a pronucleus of a fertilized oocyte. This technique is widely used in mice but remains relatively inefficient and technically demanding in other animal species. The transgene can also be introduced into one-cell-stage embryos via lentiviral infection, providing an effective alternative to standard pronuclear injections, especially in species or strains with a more challenging embryo structure. In this approach, a suspension that contains lentiviral vectors is injected into the perivitelline space of a fertilized rat embryo, which is technically less demanding and has a higher success rate. Lentiviral vectors were shown to efficiently incorporate the transgene into the genome to determine the generation of stable transgenic lines. Despite some limitations (e.g., Biosafety Level 2 requirements, DNA fragment size limits), lentiviral transgenesis is a rapid and efficient transgenesis method. Additionally, using female rats that are mated with a fertile male strain with a different dominant fur color is presented as an alternative to generate pseudopregnant foster mothers.


Assuntos
Vetores Genéticos/genética , Lentivirus/genética , Animais , Camundongos , Ratos , Ratos Transgênicos
17.
J Vis Exp ; (159)2020 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-32449724

RESUMO

Custom designed endonucleases, such as RNA-guided Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9, enable efficient genome editing in mammalian cells. Here we describe detailed procedures to seamlessly genome edit the hepatocyte nuclear factor 4 alpha (HNF4α) locus as an example in human pluripotent stem cells. Combining a piggyBac-based donor plasmid and the CRISPR-Cas9 nickase mutant in a two-step genetic selection, we demonstrate correct and efficient targeting of the HNF4α locus.


Assuntos
Edição de Genes/métodos , Células-Tronco Pluripotentes/metabolismo , Mutação Puntual/genética , Sequência de Bases , Sistemas CRISPR-Cas/genética , Elementos de DNA Transponíveis/genética , Vetores Genéticos/genética , Genoma Humano , Humanos
18.
Virology ; 546: 127-132, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32452411

RESUMO

Adeno-associated virus (AAV) is one of the most researched, clinically utilized gene therapy vectors. Though clinical success has been achieved, transgene delivery and expression may be hindered by cellular and tissue barriers. Understanding the role of receptor binding, entry, endosomal escape, cytoplasmic and nuclear trafficking, capsid uncoating, and viral transcription in therapeutic efficacy is paramount. Previous studies have shown that N-terminal regions of the AAV capsid proteins are responsible for endosomal escape and nuclear trafficking, however the mechanisms remain unknown. We identified a highly-conserved three-residue serine/threonine (S/T) motif in the capsid N-terminus, previously uncharacterized in its role in intracellular trafficking and transduction. Using alanine scanning mutagenesis, we found S155 and the flanking residues, D154 and G158, are essential for AAV2 transduction efficiency. Remarkably, specific capsid mutants show a 5 to 9-fold decrease in viral mRNA transcripts, highlighting a potential role of the S/T motif in transcription of the viral genome.


Assuntos
Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Dependovirus/genética , Regulação Viral da Expressão Gênica , Motivos de Aminoácidos , Proteínas do Capsídeo/genética , Dependovirus/química , Dependovirus/fisiologia , Vetores Genéticos/química , Vetores Genéticos/genética , Vetores Genéticos/fisiologia , Humanos , Montagem de Vírus , Replicação Viral
19.
Mem Inst Oswaldo Cruz ; 115: e190347, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32428188

RESUMO

BACKGROUND Bacillus Calmette-Guérin (BCG) is considered a promising live bacterial delivery system. However, several proposals for rBCG vaccines have not progressed, mainly due to the limitations of the available expression systems. OBJECTIVES To obtain a set of mycobacterial vectors using a range of promoters with different strengths based on a standard backbone, previously shown to be stable. METHODS Mycobacterial expression vectors based on the pLA71 vector as backbone, were obtained inserting different promoters (PAN, PαAg, PHsp60, PBlaF* and PL5) and the green fluorescence protein (GFP) as reporter gene, to evaluate features such as their relative strengths, and the in vitro (inside macrophages) and in vivo stability. FINDINGS The relative fluorescence observed with the different vectors showed increasing strength of the promoters: PAN was the weakest in both Mycobacterium smegmatis and BCG and PBlaF* was higher than PHsp60 in BCG. The relative fluorescence observed in a macrophage cell line showed that PBlaF* and PHsp60 were comparable. It was not possible to obtain strains transformed with the extrachromosomal expression vector containing the PL5 in either species. MAIN CONCLUSION We have obtained a set of potentially stable mycobacterial vectors with a arrange of expression levels, to be used in the development of rBCG vaccines.


Assuntos
Vacina BCG/imunologia , Escherichia coli/imunologia , Vetores Genéticos/imunologia , Proteínas de Fluorescência Verde/imunologia , Mycobacterium bovis/imunologia , Mycobacterium smegmatis/imunologia , Animais , Escherichia coli/genética , Feminino , Vetores Genéticos/genética , Camundongos , Camundongos Endogâmicos BALB C
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA