Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 8.311
Filtrar
1.
Molecules ; 26(10)2021 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-34067773

RESUMO

BACKGROUND: DNA-RNA compounds have shown promising protection against cell oxidative stress. This study aimed to assess the cytotoxicity, protective, or preventive effect of different experimental formulations on oral epithelia's oxidative stress in vitro. METHODS: Reconstituted human oral epithelia (RHOE) were grown air-lifted in a continuous-flow bioreactor. Mouthwashes and gels containing DNA-RNA compounds and other bioactive molecules were tested on a model of oxidative stress generated by hydrogen peroxide treatment. Epithelia viability was evaluated using a biochemical MTT-based assay and confocal microscopy; structural and ultrastructural morphology was evaluated by light microscopy and TEM. RESULTS: DNA-RNA showed non-cytotoxic activity and effectively protected against oxidative stress, but did not help in its prevention. Gel formulations did not express adequate activity compared to the mouthwashes. Excipients played a fundamental role in enhancing or even decreasing the bioactive molecules' effect. CONCLUSION: A mouthwash formulation with hydrolyzed DNA-RNA effectively protected against oxidative stress without additional enhancement by other bioactive molecules. Active compounds, such as hyaluronic acid, ß-Glucan, allantoin, bisabolol, ruscogenin, and essential oils, showed a protective effect against oxidative stress, which was not synergistic with the one of DNA-RNA. Incorporation of surfactant agents showed a reduced, yet significant, cytotoxic effect.


Assuntos
Mucosa Bucal/metabolismo , Antissépticos Bucais/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Reatores Biológicos/microbiologia , DNA/farmacologia , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Géis/farmacologia , Engenharia Genética/métodos , Humanos , Mucosa Bucal/efeitos dos fármacos , Antissépticos Bucais/metabolismo , RNA/farmacologia
2.
Methods Mol Biol ; 2262: 323-334, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33977487

RESUMO

Oncogenic KRAS mutations are common in colorectal cancer (CRC), found in ~50% of tumors, and are associated with poor prognosis and resistance to therapy. There is substantial diversity of KRAS mutations observed in CRC. Importantly, emerging clinical and experimental analysis of relatively common KRAS mutations at amino acids G12, G13, A146, and Q61 suggest that each mutation differently influences the clinical properties of a disease and response to therapy. Although clinical evidence suggests biological differences between mutant KRAS alleles, these differences and the mechanisms underlying them are not well understood, and further exploration of allele-specific differences may provide evidence for individualized therapeutics. One approach to study allelic variation involves the use of isogenic cell lines that express different endogenous KRAS mutants. Here we developed an assay using fluorescent co-selection for CRISPR-driven gene editing to generate various Kras mutations in an isogenic murine colon epithelial cell line background. This assay involves generation of a cell line stably expressing Cas9 linked to BFP and simultaneous introduction of single-guide RNAs (sgRNAs) to two different gene loci resulting in double-editing events. Single-stranded donor oligonucleotides are introduced for a GFP gene and a Kras mutant allele of our choice as templates for homologous recombination (HDR). Cells that successfully undergo HDR are GFP-positive and have a higher probability of containing the desired Kras mutation. Therefore, selection for GFP-positive cells allows us to identify those with phenotypically silent Kras edits. Ultimately, this method allows us to toggle between different mutant alleles and preserve the wild-type allele while maintaining an isogenic background.


Assuntos
Colo/metabolismo , Células Epiteliais/metabolismo , Edição de Genes , Engenharia Genética/métodos , Mutação , Proteínas Proto-Oncogênicas p21(ras)/genética , Alelos , Animais , Sistemas CRISPR-Cas , Camundongos , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores
3.
Nat Commun ; 12(1): 2689, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33976154

RESUMO

A central goal of synthetic biology is to predictably and efficiently reprogram living systems to perform computations and carry out specific biological tasks. Although there have been many advances in the bio-computational design of living systems, these advances have mainly been applied to microorganisms or cell lines; programming animal physiology remains challenging for synthetic biology because of the system complexity. Here, we present a bacteria-animal symbiont system in which engineered bacteria recognize external signals and modulate animal gene expression, twitching phenotype, and fat metabolism through RNA interference toward gfp, sbp-1, and unc-22 gene in C. elegans. By using genetic circuits in bacteria to control these RNA expressions, we are able to program the physiology of the model animal Caenorhabditis elegans with logic gates. We anticipate that engineered bacteria can be used more extensively to program animal physiology for agricultural, therapeutic, and basic science applications.


Assuntos
Caenorhabditis elegans/genética , Escherichia coli/genética , Regulação da Expressão Gênica , Redes Reguladoras de Genes/genética , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/microbiologia , Escherichia coli/fisiologia , Engenharia Genética/métodos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Microscopia de Fluorescência , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Biologia Sintética/métodos
4.
Nat Commun ; 12(1): 2770, 2021 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-33986266

RESUMO

CRISPR-based transcriptional activation is a powerful tool for functional gene interrogation; however, delivery difficulties have limited its applications in vivo. Here, we created a mouse model expressing all components of the CRISPR-Cas9 guide RNA-directed Synergistic Activation Mediator (SAM) from a single transcript that is capable of activating target genes in a tissue-specific manner. We optimized Lipid Nanoparticles and Adeno-Associated Virus guide RNA delivery approaches to achieve expression modulation of one or more genes in vivo. We utilized the SAM mouse model to generate a hypercholesteremia disease state that we could bidirectionally modulate with various guide RNAs. Additionally, we applied SAM to optimize gene expression in a humanized Transthyretin mouse model to recapitulate human expression levels. These results demonstrate that the SAM gene activation platform can facilitate in vivo research and drug discovery.


Assuntos
Sistemas CRISPR-Cas/genética , Hipercolesterolemia/genética , Lipossomos/farmacologia , Pré-Albumina/metabolismo , Ativação Transcricional/genética , Animais , Linhagem Celular , Expressão Gênica/genética , Regulação da Expressão Gênica/genética , Engenharia Genética/métodos , Células HEK293 , Humanos , Hipercolesterolemia/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Nanopartículas , Pré-Albumina/genética , RNA Guia/genética , RNA Guia/metabolismo
5.
Nat Commun ; 12(1): 2638, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33976152

RESUMO

Tepary bean (Phaseolus acutifolis A. Gray), native to the Sonoran Desert, is highly adapted to heat and drought. It is a sister species of common bean (Phaseolus vulgaris L.), the most important legume protein source for direct human consumption, and whose production is threatened by climate change. Here, we report on the tepary genome including exploration of possible mechanisms for resilience to moderate heat stress and a reduced disease resistance gene repertoire, consistent with adaptation to arid and hot environments. Extensive collinearity and shared gene content among these Phaseolus species will facilitate engineering climate adaptation in common bean, a key food security crop, and accelerate tepary bean improvement.


Assuntos
Aclimatação/genética , Evolução Molecular , Genoma de Planta , Phaseolus/genética , Melhoramento Vegetal/métodos , Mudança Climática , Produtos Agrícolas/genética , Domesticação , Secas , Segurança Alimentar , Engenharia Genética/métodos , Resposta ao Choque Térmico/genética
6.
Nat Commun ; 12(1): 2711, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33976199

RESUMO

Temporal modulation of the expression of multiple genes underlies complex complex biological phenomena. However, there are few scalable and generalizable gene circuit architectures for the programming of sequential genetic perturbations. Here, we describe a modular recombinase-based gene circuit architecture, comprising tandem gene perturbation cassettes (GPCs), that enables the sequential expression of multiple genes in a defined temporal order by alternating treatment with just two orthogonal ligands. We use tandem GPCs to sequentially express single-guide RNAs to encode transcriptional cascades that trigger the sequential accumulation of mutations. We build an all-in-one gene circuit that sequentially edits genomic loci, synchronizes cells at a specific stage within a gene expression cascade, and deletes itself for safety. Tandem GPCs offer a multi-tiered cellular programming tool for modeling multi-stage genetic changes, such as tumorigenesis and cellular differentiation.


Assuntos
Edição de Genes/métodos , Redes Reguladoras de Genes , Engenharia Genética/métodos , Genoma Humano , Plasmídeos/metabolismo , Transposases/genética , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , Carcinogênese/genética , Carcinogênese/metabolismo , Diferenciação Celular , Loci Gênicos , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação , Plasmídeos/química , RNA Guia/genética , RNA Guia/metabolismo , Transcrição Genética , Transposases/metabolismo
7.
Methods Mol Biol ; 2296: 91-141, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33977444

RESUMO

Bacteria of the genus Streptomyces are one of the most important producers of biologically active natural products. Recent robust genomic sequencing of Streptomyces strains has shown enormous genetic potential for new natural products. However, many biosynthetic gene clusters are silent. Therefore, efficient and stable genome modification methods are needed to induce their production or to manipulate them for the production of new compounds or biotechnologically improved strains. We have recently developed a simple and efficient markerless genome modification system for these bacteria based on the positive selection of double crossovers using the blue pigment indigoidine bpsA gene. This chapter is an attempt to provide methodological details of this strategy for stable markerless genomic engineering (deletions/insertions) to improve their biotechnological properties and to produce biologically active compounds.


Assuntos
Engenharia Genética/métodos , Genômica/métodos , Streptomyces/genética , Proteínas de Bactérias/genética , Produtos Biológicos , Família Multigênica/genética , Piperidonas/metabolismo
8.
Methods Mol Biol ; 2296: 185-194, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33977448

RESUMO

Electroporation is a method for the introduction of molecules (usually nucleic acids) into a cell, consisting of submitting the cells to high-voltage and short electric pulses in the presence of the exogenous DNA/molecule. It is a versatile method, adaptable to different types of cells, from bacteria to cultured cells to higher eukaryotes, and thus has applications in many diverse fields, such as environmental biology, biotechnology, genetic engineering, and medicine. Electroporation has some advantages over other genetic transformation strategies, including the simplicity of the method, a wide range of adjustable parameters (possibility of optimization), high reproducibility and avoidance of the use of chemicals toxic to cells. Here we describe an optimized electroporation procedure for the industrially important fungus Acremonium chrysogenum, using germinated conidia and fragmented young mycelium. In both cases, the transformation efficiency was higher compared to the conventional polyethylene glycol (PEG)-mediated transformation of protoplasts.


Assuntos
Eletroporação/métodos , Fungos/genética , Acremonium/genética , Biotecnologia/métodos , Engenharia Genética/métodos , Micélio/genética , Polietilenoglicóis/química , Protoplastos , Reprodutibilidade dos Testes , Transformação Genética/genética
9.
Methods Mol Biol ; 2296: 351-363, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33977458

RESUMO

Daptomycin is a cyclic lipopeptide antibiotic with potent activity against gram-positive bacteria. It has a calcium-dependent mechanism of action that disrupts multiple features of the bacterial membrane function. This antibiotic is highly demanded due to its effectiveness against to microorganisms resistant to other antibiotics, including vancomycin-resistant Staphylococcus aureus (VRSA) and methicillin-resistant S. aureus (MRSA). Daptomycin is produced by fermentation of Streptomyces roseosporus, currently identified as Streptomyces filamentosus. However, low fermentation yields and high production costs are reported. This chapter describes a method of strain improvement involving random mutagenesis, rational screening by bioassay, and flask fermentation. The ultimate objective is to select mutants of S. roseosporus overproducing daptomycin in order to design a more cost-effective daptomycin production.


Assuntos
Daptomicina/biossíntese , Streptomyces/metabolismo , Antibacterianos/biossíntese , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Daptomicina/farmacologia , Fermentação/fisiologia , Engenharia Genética/métodos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/genética , Mutagênese/genética , Streptomyces/efeitos dos fármacos , Streptomyces/genética
10.
Methods Mol Biol ; 2290: 171-185, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34009590

RESUMO

The production of biofuels from plant biomass is dependent on the availability of enzymes that can hydrolyze the plant cell wall polysaccharides to their monosaccharides. These enzyme mixtures are formed by microorganisms but their native compositions and properties are often not ideal for application. Genetic engineering of these microorganisms is therefore necessary, in which introduction of DNA is an essential precondition. The filamentous fungus Trichoderma reesei-the main producer of plant-cell-wall-degrading enzymes for biofuels and other industries-has been subjected to intensive genetic engineering toward this goal and has become one of the iconic examples of the successful genetic improvement of fungi. However, the genetic manipulation of other enzyme-producing Trichoderma species is frequently less efficient and, therefore, rarely managed. In this chapter, we therefore describe the two potent methods of Trichoderma transformation mediated by either (a) polyethylene glycol (PEG) or (b) Agrobacterium. The methods are optimized for T. reesei but can also be applied for such transformation-resilient species as T. harzianum and T. guizhouense, which are putative upcoming alternatives for T. reesei in this field. The protocols are simple, do not require extensive training or special equipment, and can be further adjusted for T. reesei mutants with particular properties.


Assuntos
Engenharia Genética/métodos , Transformação Genética/genética , Trichoderma/genética , Biocombustíveis , Biomassa , Celulase/genética , Celulose/genética , Hidrólise , Monossacarídeos/genética , Plantas/química , Plantas/metabolismo , Trichoderma/metabolismo
11.
Nat Protoc ; 16(7): 3210-3240, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33846629

RESUMO

Receptor targeting of vector particles is a key technology to enable cell type-specific in vivo gene delivery. For example, T cells in humanized mouse models can be modified by lentiviral vectors (LVs) targeted to human T-cell markers to enable them to express chimeric antigen receptors (CARs). Here, we provide detailed protocols for the generation of CD4- and CD8-targeted LVs (which takes ~9 d in total). We also describe how to humanize immunodeficient mice with hematopoietic stem cells (which takes 12-16 weeks) and precondition (over 5 d) and administer the vector stocks. Conversion of the targeted cell type is monitored by PCR and flow cytometry of blood samples. A few weeks after administration, ~10% of the targeted T-cell subtype can be expected to have converted to CAR T cells. By closely following the protocol, sufficient vector stock for the genetic manipulation of 10-15 humanized mice is obtained. We also discuss how the protocol can be easily adapted to use LVs targeted to other types of receptors and/or for delivery of other genes of interest.


Assuntos
Engenharia Genética/métodos , Linfócitos T/metabolismo , Animais , Antígenos CD/metabolismo , Células HEK293 , Humanos , Lentivirus/genética , Camundongos , Modelos Animais , Receptores de Antígenos Quiméricos/genética
12.
Int J Mol Sci ; 22(9)2021 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-33925231

RESUMO

Genome-wide transcriptomic data obtained in RNA-seq experiments can serve as a reliable source for identification of novel regulatory elements such as riboswitches and promoters. Riboswitches are parts of the 5' untranslated region of mRNA molecules that can specifically bind various metabolites and control gene expression. For that reason, they have become an attractive tool for engineering biological systems, especially for the regulation of metabolic fluxes in industrial microorganisms. Promoters in the genomes of prokaryotes are located upstream of transcription start sites and their sequences are easily identifiable based on the primary transcriptome data. Bacillus methanolicus MGA3 is a candidate for use as an industrial workhorse in methanol-based bioprocesses and its metabolism has been studied in systems biology approaches in recent years, including transcriptome characterization through RNA-seq. Here, we identify a putative lysine riboswitch in B. methanolicus, and test and characterize it. We also select and experimentally verify 10 putative B. methanolicus-derived promoters differing in their predicted strength and present their functionality in combination with the lysine riboswitch. We further explore the potential of a B. subtilis-derived purine riboswitch for regulation of gene expression in the thermophilic B. methanolicus, establishing a novel tool for inducible gene expression in this bacterium.


Assuntos
Bacillus/genética , Engenharia Genética/métodos , Riboswitch/genética , Bacillus/metabolismo , Proteínas de Bactérias/metabolismo , Biologia Computacional/métodos , Genoma Bacteriano/genética , Análise do Fluxo Metabólico/métodos , Regiões Promotoras Genéticas/genética , Sequências Reguladoras de Ácido Nucleico , Sítio de Iniciação de Transcrição/fisiologia , Transcriptoma/genética
13.
Int J Mol Sci ; 22(7)2021 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-33805113

RESUMO

According to Darwin's theory, endless evolution leads to a revolution. One such example is the Clustered Regularly Interspaced Palindromic Repeats (CRISPR)-Cas system, an adaptive immunity system in most archaea and many bacteria. Gene editing technology possesses a crucial potential to dramatically impact miscellaneous areas of life, and CRISPR-Cas represents the most suitable strategy. The system has ignited a revolution in the field of genetic engineering. The ease, precision, affordability of this system is akin to a Midas touch for researchers editing genomes. Undoubtedly, the applications of this system are endless. The CRISPR-Cas system is extensively employed in the treatment of infectious and genetic diseases, in metabolic disorders, in curing cancer, in developing sustainable methods for fuel production and chemicals, in improving the quality and quantity of food crops, and thus in catering to global food demands. Future applications of CRISPR-Cas will provide benefits for everyone and will save countless lives. The technology is evolving rapidly; therefore, an overview of continuous improvement is important. In this review, we aim to elucidate the current state of the CRISPR-Cas revolution in a tailor-made format from its discovery to exciting breakthroughs at the application level and further upcoming trends related to opportunities and challenges including ethical concerns.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Engenharia Genética/métodos , Animais , Archaea/metabolismo , Bactérias/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Produtos Agrícolas/genética , Engenharia Genética/história , Genoma , História do Século XX , História do Século XXI , Humanos , Gado
14.
J Plant Physiol ; 261: 153411, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33872932

RESUMO

Plant transformation remains the most sought-after technology for functional genomics and crop genetic improvement, especially for introducing specific new traits and to modify or recombine already existing traits. Along with many other agricultural technologies, the global production of genetically engineered crops has steadily grown since they were first introduced 25 years ago. Since the first transfer of DNA into plant cells using Agrobacterium tumefaciens, different transformation methods have enabled rapid advances in molecular breeding approaches to bring crop varieties with novel traits to the market that would be difficult or not possible to achieve with conventional breeding methods. Today, transformation to produce genetically engineered crops is the fastest and most widely adopted technology in agriculture. The rapidly increasing number of sequenced plant genomes and information from functional genomics data to understand gene function, together with novel gene cloning and tissue culture methods, is further accelerating crop improvement and trait development. These advances are welcome and needed to make crops more resilient to climate change and to secure their yield for feeding the increasing human population. Despite the success, transformation remains a bottleneck because many plant species and crop genotypes are recalcitrant to established tissue culture and regeneration conditions, or they show poor transformability. Improvements are possible using morphogenetic transcriptional regulators, but their broader applicability remains to be tested. Advances in genome editing techniques and direct, non-tissue culture-based transformation methods offer alternative approaches to enhance varietal development in other recalcitrant crops. Here, we review recent developments in plant transformation and regeneration, and discuss opportunities for new breeding technologies in agriculture.


Assuntos
Produtos Agrícolas/genética , Engenharia Genética/métodos , Melhoramento Vegetal/métodos , Plantas Geneticamente Modificadas/genética
15.
Int J Mol Sci ; 22(7)2021 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-33807162

RESUMO

Prader-Willi syndrome (PWS) is a neurogenetic multifactorial disorder caused by the deletion or inactivation of paternally imprinted genes on human chromosome 15q11-q13. The affected homologous locus is on mouse chromosome 7C. The positional conservation and organization of genes including the imprinting pattern between mice and men implies similar physiological functions of this locus. Therefore, considerable efforts to recreate the pathogenesis of PWS have been accomplished in mouse models. We provide a summary of different mouse models that were generated for the analysis of PWS and discuss their impact on our current understanding of corresponding genes, their putative functions and the pathogenesis of PWS. Murine models of PWS unveiled the contribution of each affected gene to this multi-facetted disease, and also enabled the establishment of the minimal critical genomic region (PWScr) responsible for core symptoms, highlighting the importance of non-protein coding genes in the PWS locus. Although the underlying disease-causing mechanisms of PWS remain widely unresolved and existing mouse models do not fully capture the entire spectrum of the human PWS disorder, continuous improvements of genetically engineered mouse models have proven to be very powerful and valuable tools in PWS research.


Assuntos
Modelos Animais de Doenças , Síndrome de Prader-Willi/genética , Síndrome de Prader-Willi/metabolismo , Animais , Mapeamento Cromossômico/métodos , Metilação de DNA , Engenharia Genética/métodos , Genoma , Impressão Genômica , Humanos , Masculino , Camundongos , RNA Nucleolar Pequeno/genética
16.
Nat Protoc ; 16(5): 2345-2380, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33903757

RESUMO

We previously developed REXER (Replicon EXcision Enhanced Recombination); this method enables the replacement of >100 kb of the Escherichia coli genome with synthetic DNA in a single step and allows the rapid identification of non-viable or otherwise problematic sequences with nucleotide resolution. Iterative repetition of REXER (GENESIS, GENomE Stepwise Interchange Synthesis) enables stepwise replacement of longer contiguous sections of genomic DNA with synthetic DNA, and even the replacement of the entire E. coli genome with synthetic DNA. Here we detail protocols for REXER and GENESIS. A standard REXER protocol typically takes 7-10 days to complete. Our description encompasses (i) synthetic DNA design, (ii) assembly of synthetic DNA constructs, (iii) utilization of CRISPR-Cas9 coupled to lambda-red recombination and positive/negative selection to enable the high-fidelity replacement of genomic DNA with synthetic DNA (or insertion of synthetic DNA), (iv) evaluation of the success of the integration and replacement and (v) identification of non-tolerated synthetic DNA sequences with nucleotide resolution. This protocol provides a set of precise genome engineering methods to create custom synthetic E. coli genomes.


Assuntos
Escherichia coli/genética , Engenharia Genética/métodos , Genômica/métodos , DNA Bacteriano/genética , Genoma Bacteriano/genética , Recombinação Genética
17.
Neuron ; 109(7): 1080-1083, 2021 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-33831364

RESUMO

The iPSC Neurodegenerative Disease Initiative (iNDI) is the largest-ever iPSC genome engineering project. iNDI will model more than 100 mutations associated with Alzheimer's disease and related dementias (ADRD) in isogenic iPSC lines. Resulting cell lines and phenotypic datasets will be broadly shared.


Assuntos
Engenharia Genética/métodos , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/terapia , Pesquisa com Células-Tronco , Transplante de Células-Tronco , Doença de Alzheimer/terapia , Humanos , Células-Tronco Pluripotentes Induzidas/transplante , Células-Tronco Neurais/transplante
18.
mBio ; 12(2)2021 03 02.
Artigo em Inglês | MEDLINE | ID: covidwho-1115089

RESUMO

There are no approved vaccines against the life-threatening Middle East respiratory syndrome coronavirus (MERS-CoV). Attenuated vaccines have proven their potential to induce strong and long-lasting immune responses. We have previously described that severe acute respiratory syndrome coronavirus (SARS-CoV) envelope (E) protein is a virulence factor. Based on this knowledge, a collection of mutants carrying partial deletions spanning the C-terminal domain of the E protein (rMERS-CoV-E*) has been generated using a reverse genetics system. One of these mutants, MERS-CoV-E*Δ2in, was attenuated and provided full protection in a challenge with virulent MERS-CoV after a single immunization dose. The MERS-CoV-E*Δ2in mutant was stable as it maintained its attenuation after 16 passages in cell cultures and has been selected as a promising vaccine candidate.IMPORTANCE The emergence of the new highly pathogenic human coronavirus SARS-CoV-2 that has already infected more than 80 million persons, killing nearly two million of them, clearly indicates the need to design efficient and safe vaccines protecting from these coronaviruses. Modern vaccines can be derived from virus-host interaction research directed to the identification of signaling pathways essential for virus replication and for virus-induced pathogenesis, in order to learn how to attenuate these viruses and design vaccines. Using a reverse genetics system developed in our laboratory, an infectious cDNA clone of MERS-CoV was engineered. Using this cDNA, we sequentially deleted several predicted and conserved motifs within the envelope (E) protein of MERS-CoV, previously associated with the presence of virulence factors. The in vitro and in vivo evaluation of these deletion mutants highlighted the relevance of predicted linear motifs in viral pathogenesis. Two of them, an Atg8 protein binding motif (Atg8-BM), and a forkhead-associated binding motif (FHA-BM), when deleted, rendered an attenuated virus that was evaluated as a vaccine candidate, leading to full protection against challenge with a lethal dose of MERS-CoV. This approach can be extended to the engineering of vaccines protecting against the new pandemic SARS-CoV-2.


Assuntos
Coronavírus da Síndrome Respiratória do Oriente Médio/patogenicidade , COVID-19/imunologia , COVID-19/prevenção & controle , Engenharia Genética/métodos , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Vacinas Atenuadas/uso terapêutico , Vacinas Virais/uso terapêutico
19.
Int J Mol Sci ; 22(4)2021 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-33669386

RESUMO

Animal models are crucial to understanding human disease biology and developing new therapies. By far the most common animal used to investigate prevailing questions about human disease is the mouse. Mouse models are powerful tools for research as their small size, limited lifespan, and defined genetic background allow researchers to easily manipulate their genome and maintain large numbers of animals in general laboratory spaces. However, it is precisely these attributes that make them so different from humans and explains, in part, why these models do not accurately predict drug responses in human patients. This is particularly true of the neurofibromatoses (NFs), a group of genetic diseases that predispose individuals to tumors of the nervous system, the most common of which is Neurofibromatosis type 1 (NF1). Despite years of research, there are still many unanswered questions and few effective treatments for NF1. Genetically engineered mice have drastically improved our understanding of many aspects of NF1, but they do not exemplify the overall complexity of the disease and some findings do not translate well to humans due to differences in body size and physiology. Moreover, NF1 mouse models are heavily reliant on the Cre-Lox system, which does not accurately reflect the molecular mechanism of spontaneous loss of heterozygosity that accompanies human tumor development. Spontaneous and genetically engineered large animal models may provide a valuable supplement to rodent studies for NF1. Naturally occurring comparative models of disease are an attractive prospect because they occur on heterogeneous genetic backgrounds and are due to spontaneous rather than engineered mutations. The use of animals with naturally occurring disease has been effective for studying osteosarcoma, lymphoma, and diabetes. Spontaneous NF-like symptoms including neurofibromas and malignant peripheral nerve sheath tumors (MPNST) have been documented in several large animal species and share biological and clinical similarities with human NF1. These animals could provide additional insight into the complex biology of NF1 and potentially provide a platform for pre-clinical trials. Additionally, genetically engineered porcine models of NF1 have recently been developed and display a variety of clinical features similar to those seen in NF1 patients. Their large size and relatively long lifespan allow for longitudinal imaging studies and evaluation of innovative surgical techniques using human equipment. Greater genetic, anatomic, and physiologic similarities to humans enable the engineering of precise disease alleles found in human patients and make them ideal for preclinical pharmacokinetic and pharmacodynamic studies of small molecule, cellular, and gene therapies prior to clinical trials in patients. Comparative genomic studies between humans and animals with naturally occurring disease, as well as preclinical studies in large animal disease models, may help identify new targets for therapeutic intervention and expedite the translation of new therapies. In this review, we discuss new genetically engineered large animal models of NF1 and cases of spontaneous NF-like manifestations in large animals, with a special emphasis on how these comparative models could act as a crucial translational intermediary between specialized murine models and NF1 patients.


Assuntos
Animais Geneticamente Modificados , Modelos Animais de Doenças , Engenharia Genética/métodos , Acúmulo de Mutações , Neurofibromatose 1/genética , Animais , Bovinos , Cães , Humanos , Camundongos , Terapia de Alvo Molecular/métodos , Neurofibromatose 1/tratamento farmacológico , Suínos/genética
20.
Mol Genet Genomics ; 296(3): 485-500, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33751237

RESUMO

Finding and explaining the functions of genes in plants have promising applications in crop improvement and bioprospecting and hence, it is important to compare various techniques available for gene function identification in plants. Today, the most popular technology among researchers to identify the functions of genes is the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9)-based genome editing method. But by no means can we say that CRISPR/Cas9 is the go-to method for all purposes. It comes with its own baggage. Researchers will agree and have lived through at least seven more technologies deployed to find the functions of genes, which come under three umbrellas: 1. genetic engineering, 2. transient expression, and 3. chemical/physical mutagenesis. Each of the methods evolved when the previous one ran into an insurmountable problem. In this review, we compare the eight technologies against one another on 14 parameters. This review lays bare the pros and cons, and similarities and dissimilarities of various methods. Every method comes with its advantages and disadvantages. For example, the CRISPR/Cas9-based genome editing is an excellent method for modifying gene sequences, creating allelic versions of genes, thereby aiding the understanding of gene function. But it comes with the baggage of unwanted or off-target mutations. Then, we have methods based on random or targeted knockout of the gene, knockdown, and overexpression of the gene. Targeted disruption of genes is required for complete knockout of gene function, which may not be accomplished by editing. We have also discussed the strategies to overcome the shortcomings of the targeted gene-knockout and the CRISPR/Cas9-based methods. This review serves as a comprehensive guide towards the understanding and comparison of various technologies available for gene function identification in plants and hence, it will find application for crop improvement and bioprospecting related research.


Assuntos
Edição de Genes/métodos , Engenharia Genética/métodos , Plantas/genética , Animais , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Genoma de Planta/genética , Mutagênese/genética , Mutação/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...