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1.
World J Microbiol Biotechnol ; 35(7): 111, 2019 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-31280424

RESUMO

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) immune systems in bacteria have been used as tools for genome engineering. Thus far, the CRISPR-Cas system has been used in various yeast, bacterial, and mammalian cells. Saccharomyces cerevisiae is a nonpathogenic yeast, classified under "generally recognized as safe", and has long been used to produce consumables such as alcohol or bread. Additionally, recombinant cells of S. cerevisiae have been constructed and used to produce various bio-based chemicals. Some types of CRISPR-Cas system for genetic manipulation have been constructed during the early developmental stages of the CRISPR-Cas system and have been mainly used for gene knock-in and knock-out manipulations. Thereafter, these systems have been used for various novel purposes such as metabolic engineering and tolerance engineering. In this review, we have summarized different aspects of the CRISPR-Cas in the yeast S. cerevisiae, from its basic principles to various applications. This review describes the CRISPR system in S. cerevisiae based on the differences in its origin and efficiency followed by its basic applications; for example, its involvement in gene knock-in and knock-out has been outlined. Finally, advanced applications of the CRISPR system in the bioproduction of useful chemicals have been summarized.


Assuntos
Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Engenharia Metabólica/métodos , Saccharomyces cerevisiae/metabolismo , Edição de Genes/métodos , Regulação Fúngica da Expressão Gênica , Técnicas de Introdução de Genes/métodos , Técnicas de Inativação de Genes/métodos , Saccharomyces cerevisiae/genética
2.
Dev Growth Differ ; 61(4): 265-275, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31037730

RESUMO

The CRISPR-Cas9 technology has been a powerful means to manipulate the genome in a wide range of organisms. A series of GFP knocked-in (GFPKI ) Drosophila strains have been generated through CRISPR-Cas9-induced double strand breaks coupled with homology-directed repairs in the presence of donor plasmids. They visualized specific cell types or intracellular structures in both fixed and live specimen. We provide a rapid and efficient strategy to identify KI lines. This method requires neither co-integration of a selection marker nor prior establishment of sgRNA-expressing transgenic lines. The injection of the mixture of a sgRNA/Cas9 expression plasmid and a donor plasmid into cleavage stage embryos efficiently generated multiple independent KI lines. A PCR-based selection allows to identify KI fly lines at the F1 generation (approximately 4 weeks after injection). These GFPKI strains have been deposited in the Kyoto Drosophila stock center, and made freely available to researchers at non-profit organizations. Thus, they will be useful resources for Drosophila research.


Assuntos
Sistemas CRISPR-Cas/genética , Drosophila/genética , Edição de Genes/métodos , Técnicas de Introdução de Genes/métodos , Proteínas de Fluorescência Verde/genética , Animais , Fatores de Tempo
3.
Bioengineered ; 10(1): 98-107, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31023186

RESUMO

Progranulin has multiple functions in several physiological and pathological processes, including embryonic development, wound repair, tumorigenesis, inflammation and neurodegeneration. To investigate the transcriptional regulation of the PGRN gene, a luciferase knock-in reporter system was established in HEK293 cells by integrating luciferase gene in the genome controlled by the endogenous PGRN promoter using CRISPR/Cas9. PCR results demonstrated the site-specific integration of the exogenous luciferase gene into the genome. To validate the novel luciferase knock-in system, a CRISPR/Cas9 transcription activation/repression system for the PGRN gene was constructed and applied to the knock-in system. In addition, phorbol ester (phorbol 12-myristate, 13-acetate), previously reported as activating the expression of PGRN, was applied to the system. The results indicated that luciferase activity was directly correlated with the activity of the PGRN endogenous promoter. This novel system will be a useful tool for investigating the transcriptional regulation of PGRN, and it has great potential in screening the drugs targeting PGRN.


Assuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Efeito Fundador , Técnicas de Introdução de Genes/métodos , Luciferases/genética , Progranulinas/genética , Sequência de Bases , Proteína 9 Associada à CRISPR/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Genoma Humano , Células HEK293 , Humanos , Luciferases/metabolismo , Progranulinas/agonistas , Progranulinas/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos , Transdução de Sinais , Acetato de Tetradecanoilforbol/farmacologia , Transcrição Genética/efeitos dos fármacos
4.
Methods Mol Biol ; 1942: 61-69, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30900175

RESUMO

Human pluripotent stem cells (hPSCs) offer powerful platforms for studying mechanisms of human diseases and for evaluating potential treatments. Genome editing, particularly the CRISPR/Cas9-based method, is highly effective for generating cell and animal models to study genetic human diseases. However, the procedure for generating gene-edited hPSCs is laborious, time consuming and unintentional genetic changes may confound the consequent experiments and conclusions. Here we describe one-step knockin of the NanoLuc luciferase gene (Nluc) to the fragile X syndrome gene, FMR1, in a human embryonic stem cell line (hESC), H1, and a fragile X disease model human induced pluripotent stem cell line (hiPSC), FX-iPSC. The luciferase reporter cell lines provide new platforms for exploring potential treatments for fragile X syndrome. The shortened and scarless targeting method described here can be effectively applied to other genes.


Assuntos
Sistemas CRISPR-Cas , Proteína do X Frágil de Retardo Mental/genética , Edição de Genes/métodos , Técnicas de Introdução de Genes/métodos , Genoma Humano , Células-Tronco Pluripotentes Induzidas/metabolismo , Luciferases/metabolismo , Células Cultivadas , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Síndrome do Cromossomo X Frágil/genética , Humanos , Células-Tronco Pluripotentes Induzidas/citologia
5.
Genesis ; 57(5): e23292, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30884088

RESUMO

Medullary thyroid carcinoma (MTC) develops from hyperplasia of thyroid C cells and represents one of the major causes of thyroid cancer mortality. Mutations in the cysteine-rich domain (CRD) of the RET gene are the most prevalent genetic cause of MTC. The current consensus holds that such cysteine mutations cause ligand-independent dimerization and constitutive activation of RET. However, given the number of the CRD mutations left uncharacterized, our understanding of the pathogenetic mechanisms by which CRD mutations lead to MTC remains incomplete. We report here that RET(C618F), a mutation identified in MTC patients, displays moderately high basal activity and requires the ligand for its full activation. To assess the biological significance of RET(C618F) in organogenesis, we generated a knock-in mouse line conditionally expressing RET(C618F) cDNA by the Ret promoter. The RET(C618F) allele can be made to be Ret-null and express mCherry by Cre-loxP recombination, which allows the assessment of the biological influence of RET(C618F) in vivo. Mice expressing RET(C618F) display mild C cell hyperplasia and increased numbers of enteric neurons, indicating that RET(C618F) confers gain-of-function phenotypes. This mouse line serves as a novel biological platform for investigating pathogenetic mechanisms involved in MTC and enteric hyperganglionosis.


Assuntos
Carcinoma Neuroendócrino/genética , Proteínas Proto-Oncogênicas c-ret/genética , Neoplasias da Glândula Tireoide/genética , Animais , Carcinoma Neuroendócrino/metabolismo , Linhagem Celular Tumoral , Sistema Nervoso Entérico/metabolismo , Sistema Nervoso Entérico/patologia , Técnicas de Introdução de Genes/métodos , Mutação em Linhagem Germinativa , Humanos , Hiperplasia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Doenças do Sistema Nervoso/genética , Doenças do Sistema Nervoso/metabolismo , Doenças do Sistema Nervoso/patologia , Proteínas Proto-Oncogênicas c-ret/biossíntese , Proteínas Proto-Oncogênicas c-ret/metabolismo , Hiperplasia do Timo/genética , Hiperplasia do Timo/metabolismo , Glândula Tireoide/metabolismo , Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/metabolismo
6.
Methods Mol Biol ; 1953: 105-119, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30912018

RESUMO

Small molecule-induced targeted protein degradation is a powerful approach for drug target validation given its selectivity, high kinetic resolution, dose dependency, and reversibility. Out of the several methods that have been reported so far, the 12 kDa degradation tag (dTAG) system has the advantage of hijacking a degradation machinery that is ubiquitously expressed in all human tissues. Therefore it is independent of expressing additional, exogenous factors and additionally permits target validation in vivo. Here, we describe the protocol for generation and validation of clones harboring knock-in of a selectable dTAG cassette at the endogenous locus of proteins of interest using the near-haploid cell line KBM7.


Assuntos
Descoberta de Drogas/métodos , Técnicas de Introdução de Genes/métodos , Proteólise/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Linhagem Celular , Clonagem Molecular/métodos , Edição de Genes/métodos , Vetores Genéticos/genética , Haploidia , Humanos , Plasmídeos/genética , Proteínas/genética , Proteínas/metabolismo
7.
Methods Mol Biol ; 1953: 213-230, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30912024

RESUMO

The use of CRISPR/Cas9 to modify the mouse genome has gained immense interest in the past few years since it allows the direct modification of embryos, bypassing the need of labor-intensive procedures for the manipulation of embryonic stem cells. By shortening the overall timelines and reducing the costs for the generation of new genetically modified mouse lines (Li et al., Nat Biotechnol 31: 681-683, 2013), this technology has rapidly become a major tool for in vivo drug discovery applications.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Técnicas de Introdução de Genes/métodos , Técnicas de Inativação de Genes/métodos , Camundongos/genética , Alelos , Animais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Feminino , Genoma , Técnicas de Genotipagem/métodos , Humanos , Masculino , Camundongos/embriologia , Camundongos Endogâmicos C57BL , RNA Guia/genética
8.
Prep Biochem Biotechnol ; 49(4): 384-390, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30822257

RESUMO

Expression cell line constructed by random integration method will often meet with unstable expression problem because target genes may be integrated into unstable region of chromatin. Rational cell line construction can overcome this shortcoming by inserting target gene into stable region of chromatin specifically. Here, we successfully got one knock-in cell line where light chain and heavy chain genes of antibody was site specifically integrated into stable hot spot reported before via homologous dependent recombination method mediated by CRISPR/Cas9. The targeting efficiency was around 1.35%. This cell line together with other three pre-established targeting cell lines (targeting with glucagon-like peptide 1 with human serum albumin fusion protein gene, or NGGH) were all undergoing protein expression level detection. In adherent cell mode, the amount of antibody expressed per cell per day were all around 0.006 pg/cell/day over passage 3, 12, 23, 35 and 50 while the amount of NGGH expressed per cell per day of 3 cell lines were all around 1.2 pg/cell/day over passage 3, 12, 23, 35 and 50. In batch mode, the antibody concentration within supernatant were around 2.5 µg/L over passage 1, 25, and 50 while the NGGH fusion protein concentration within supernatant were around 17 mg/L over passage 1, 25, and 50.


Assuntos
Engenharia Celular/métodos , Técnicas de Introdução de Genes/métodos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/genética , Animais , Bevacizumab/genética , Células CHO , Sistemas CRISPR-Cas , Cricetulus , Peptídeo 1 Semelhante ao Glucagon/análise , Peptídeo 1 Semelhante ao Glucagon/genética , Humanos , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/genética , Albumina Sérica Humana/análise , Albumina Sérica Humana/genética
9.
Genetics ; 211(4): 1155-1177, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30765420

RESUMO

To understand gene function, the cre/loxP conditional system is the most powerful available for temporal and spatial control of expression in mouse. However, the research community requires more cre recombinase expressing transgenic mouse strains (cre-drivers) that restrict expression to specific cell types. To address these problems, a high-throughput method for large-scale production that produces high-quality results is necessary. Further, endogenous promoters need to be chosen that drive cell type specific expression, or we need to further focus the expression by manipulating the promoter. Here we test the suitability of using knock-ins at the docking site 5' of Hprt for rapid development of numerous cre-driver strains focused on expression in adulthood, using an improved cre tamoxifen inducible allele (icre/ERT2), and testing a novel inducible-first, constitutive-ready allele (icre/f3/ERT2/f3). In addition, we test two types of promoters either to capture an endogenous expression pattern (MaxiPromoters), or to restrict expression further using minimal promoter element(s) designed for expression in restricted cell types (MiniPromoters). We provide new cre-driver mouse strains with applicability for brain and eye research. In addition, we demonstrate the feasibility and applicability of using the locus 5' of Hprt for the rapid generation of substantial numbers of cre-driver strains. We also provide a new inducible-first constitutive-ready allele to further speed cre-driver generation. Finally, all these strains are available to the research community through The Jackson Laboratory.


Assuntos
Encéfalo/metabolismo , Olho/metabolismo , Técnicas de Introdução de Genes/métodos , Camundongos Transgênicos/genética , Tamoxifeno/farmacologia , Ativação Transcricional/efeitos dos fármacos , Animais , Efeito Fundador , Hipoxantina Fosforribosiltransferase/genética , Hipoxantina Fosforribosiltransferase/metabolismo , Integrases/genética , Integrases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas
10.
PLoS One ; 14(1): e0202377, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30615627

RESUMO

One of the most popular techniques in zebrafish research is microinjection. This is a rapid and efficient way to genetically manipulate early developing embryos, and to introduce microbes, chemical compounds, nanoparticles or tracers at larval stages. Here we demonstrate the development of a machine learning software that allows for microinjection at a trained target site in zebrafish eggs at unprecedented speed. The software is based on the open-source deep-learning library Inception v3. In a first step, the software distinguishes wells containing embryos at one-cell stage from wells to be skipped with an accuracy of 93%. A second step was developed to pinpoint the injection site. Deep learning allows to predict this location on average within 42 µm to manually annotated sites. Using a Graphics Processing Unit (GPU), both steps together take less than 100 milliseconds. We first tested our system by injecting a morpholino into the middle of the yolk and found that the automated injection efficiency is as efficient as manual injection (~ 80%). Next, we tested both CRISPR/Cas9 and DNA construct injections into the zygote and obtained a comparable efficiency to that of an experienced experimentalist. Combined with a higher throughput, this results in a higher yield. Hence, the automated injection of CRISPR/Cas9 will allow high-throughput applications to knock out and knock in relevant genes to study their mechanisms or pathways of interest in diverse areas of biomedical research.


Assuntos
Aprendizado Profundo , Embrião não Mamífero/embriologia , Desenvolvimento Embrionário/genética , Edição de Genes/métodos , Técnicas de Introdução de Genes/métodos , Peixe-Zebra , Animais , Microinjeções/métodos , Peixe-Zebra/embriologia , Peixe-Zebra/genética
11.
BMC Biotechnol ; 19(1): 7, 2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30646876

RESUMO

BACKGROUND: Non-human primate (NHP) models can closely mimic human physiological functions and are therefore highly valuable in biomedical research. Genome editing is now developing rapidly due to the precision and efficiency offered by engineered site-specific endonuclease-based systems, such as transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) system. It has been demonstrated that these programmable nucleases can introduce genetic changes in embryos from many species including NHPs. In 2014, we reported the first genetic editing of macaques using TALENs and CRISPR/Cas9. Subsequently, we characterized the phenotype of a methyl CpG binding protein 2 (MECP2)-mutant cynomolgus monkey model of Rett syndrome generated using the TALEN approach. These efforts not only accelerated the advance of modeling genetic diseases in NHPs, but also encouraged us to develop specific gene knock-in monkeys. In this study, we assess the possibility of homologous recombination (HR)-mediated gene replacement using TALENs in monkeys, and generate preimplantation embryos carrying an EmGFP fluorescent reporter constructed in the OCT4 gene. RESULT: We assembled a pair of TALENs specific to the first exon of the OCT4 gene and constructed a donor vector consisting of the homology arms cloned from the monkey genome DNA, flanking an EmGFP cassette. Next, we co-injected the TALENs-coding plasmid and donor plasmid into the cytoplasm of 122 zygotes 6-8 h after fertilization. Sequencing and immunofluorescence revealed that the OCT4-EmGFP knock-in allele had been successfully generated by TALENs-mediated HR at an efficiency of 11.3% (7 out of 62) or 11.1% (1 out of 9), respectively, in monkey embryos. CONCLUSION: We have successfully, for the first time, obtained OCT4-EmGFP knock-in monkey embryos via HR mediated by TALENs. Our results suggest that gene targeting through TALEN-assisted HR is a useful approach to introduce precise genetic modification in NHPs.


Assuntos
Edição de Genes/métodos , Técnicas de Introdução de Genes/métodos , Recombinação Homóloga , Macaca fascicularis/embriologia , Macaca fascicularis/genética , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/genética , Animais , Animais Geneticamente Modificados , Feminino , Genótipo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo
12.
Methods Mol Biol ; 1864: 295-310, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30415344

RESUMO

With ever-increasing genomic information combined with modern tools for genome modification, we are entering a new era of plant biotechnology. One major tool used for genome modification is the zinc finger nuclease (ZFN). Here, we discuss how ZFNs have proven useful in many genome modification applications. In order to remove the function of a gene or genes, targeted mutagenesis using ZFNs has been readily demonstrated creating numerous gene knockouts, and gene deletion has been demonstrated with removal of gene segments both native and transgenic up to 9 Mb. Applications for gain of function have also been demonstrated. Precision gene editing using ZFNs has resulted in the development of herbicide tolerance, and numerous forms of targeted gene addition have been exhibited. In addition to genome modification, this chapter also describes the use of zinc finger protein transcription factors (ZFP-TFs) for gene regulation in order to provide useful modification of gene expression resulting in altered phenotypes.


Assuntos
Biotecnologia/métodos , Edição de Genes/métodos , Engenharia Genética/métodos , Nucleases de Dedos de Zinco/metabolismo , Biotecnologia/instrumentação , Mutação com Ganho de Função/genética , Deleção de Genes , Edição de Genes/instrumentação , Regulação da Expressão Gênica de Plantas/genética , Técnicas de Introdução de Genes/instrumentação , Técnicas de Introdução de Genes/métodos , Engenharia Genética/instrumentação , Genoma de Planta/genética , Nucleases de Dedos de Zinco/genética
13.
Methods Mol Biol ; 1874: 115-137, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30353511

RESUMO

The rapidly evolving CRISPR/Cas9-mediated genome editing provides the convenience of genome manipulation directly in mouse zygotes for a number of genomic manipulations; but knockins of large insertions prove to be relatively inefficient at least with double-strand DNA as targeting constructs. Here, we describe an alternative approach to the direct genome editing in mouse zygotes by generating knockin alleles in mouse embryonic stem cells first with CRIPSR-mediated homologous recombination. Our results show this approach is efficient and requires no drug selection in mouse embryonic stem cells as in classic gene targeting. As the result, knockin alleles across many target loci are created in mouse embryonic stem cells and readily transmitted through germline. The knockin alleles created in ES cells can also serve as valuable tools for in vitro stem cell differentiation.


Assuntos
Técnicas de Introdução de Genes/métodos , Células Germinativas/citologia , Células-Tronco Embrionárias Murinas/citologia , Alelos , Animais , Sistemas CRISPR-Cas , Diferenciação Celular , Células Cultivadas , Recombinação Homóloga , Camundongos
14.
G3 (Bethesda) ; 9(2): 591-599, 2019 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-30591434

RESUMO

The modification of the mouse genome by site-specific gene insertion of transgenes and other genetic elements allows the study of gene function in different developmental stages and in the pathogenesis of diseases. Here, we generated a "genomic safe harbor" Hipp11 (H11) locus-specific knock-in transgenic mouse line in which the albumin promoter is used to drive the expression of the reverse tetracycline transactivator (rtTA) in the liver. The newly generated H11-albumin-rtTA transgenic mice were bred with tetracycline-operator-Histone-2B-green fluorescent protein (TetO-H2BGFP) mice to assess inducibility and tissue-specificity. Expression of the H2BGFP fusion protein was observed exclusively upon doxycycline (Dox) induction in the liver of H11-albumin-rtTA/TetO-H2BGFP double transgenic mice. To further analyze the ability of the Dox-inducible H11-albumin-rtTA mice to implement conditional DNA recombination, H11-albumin-rtTA transgenic mice were crossed with TetO-Cre and Ai14 mice to generate H11-albumin-rtTA/TetO-Cre/Ai14 triple transgenic mice. We successfully confirmed that the Cre-mediated recombination efficiency was as strong in Dox-induced H11-albumin-rtTA /TetO-Cre/Ai14 mice as in the control albumin-Cre/A14 mice. Finally, to characterize the expression-inducing effects of Dox in H11-albumin-rtTA/TetO-H2BGFP mice in detail, we examined GFP expression in embryos at different developmental stages and found that newly conceived H11-albumin-rtTA/TetO-H2BGFP embryos of Dox-treated pregnant female mice were expressing reporter GFP by E16.5. Our study demonstrates that these new H11-albumin-rtTA transgenic mice are a powerful and efficient tool for the temporally and spatially conditional manipulation of gene expression in the liver, and illustrates how genetic crosses with these new mice enable the generation of complex multi-locus transgenic animals for mechanistic studies.


Assuntos
Técnicas de Introdução de Genes/métodos , Fígado/metabolismo , Camundongos Transgênicos/genética , Albuminas/genética , Albuminas/metabolismo , Animais , Doxiciclina/farmacologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Regiões Promotoras Genéticas , Transativadores/genética , Transativadores/metabolismo , Ativação Transcricional/efeitos dos fármacos
15.
Methods Mol Biol ; 1869: 207-230, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30324526

RESUMO

To maximize the physiological relevance of in vivo brain tumor mouse models designed to study the downstream effects of oncogenic mutations, it is important to express the mutated genes at appropriate levels, in relevant cell types, and in the proper developmental context. For recurrent mutations found in the heterozygous state in tumors, expression of the mutation from the endogenous locus is a more physiologically relevant recapitulation of the brain tumor genome. Here, we describe an approach to generate knock-in mice with an inducible mutation recombined into the endogenous locus. In these engineered mice, the mutated allele is designed for expression controlled by the endogenous promoter and regulatory elements after Cre recombinase-mediated deletion of a loxP-STOP-loxP cassette inserted upstream of the translational start site. To preserve the structure of the endogenous locus, mutations or additional elements may need to be inserted at a considerable distance from the loxP-STOP-loxP cassette. We used recombineering to build a construct with two selectable markers and multiple genetic alterations that can be introduced into the endogenous allele in cis with a single ES cell targeting.


Assuntos
Neoplasias Encefálicas/genética , Técnicas de Introdução de Genes/métodos , Engenharia Genética/métodos , Loci Gênicos , Mutação/genética , Oncogenes , Animais , Cromossomos Artificiais Bacterianos/genética , Modelos Animais de Doenças , Eletroporação , Vetores Genéticos/metabolismo , Genômica , Recombinação Homóloga/genética , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Mutação Puntual/genética
16.
Hum Mol Genet ; 28(8): 1260-1273, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30535360

RESUMO

Myelin sheath thickness is precisely regulated and essential for rapid propagation of action potentials along myelinated axons. In the peripheral nervous system, extrinsic signals from the axonal protein neuregulin 1 (NRG1) type III regulate Schwann cell fate and myelination. Here we ask if modulating NRG1 type III levels in neurons would restore myelination in a model of congenital hypomyelinating neuropathy (CHN). Using a mouse model of CHN, we improved the myelination defects by early overexpression of NRG1 type III. Surprisingly, the improvement was independent from the upregulation of Egr2 or essential myelin genes. Rather, we observed the activation of MAPK/ERK and other myelin genes such as peripheral myelin protein 2 and oligodendrocyte myelin glycoprotein. We also confirmed that the permanent activation of MAPK/ERK in Schwann cells has detrimental effects on myelination. Our findings demonstrate that the modulation of axon-to-glial NRG1 type III signaling has beneficial effects and improves myelination defects during development in a model of CHN.


Assuntos
Bainha de Mielina/metabolismo , Neuregulina-1/genética , Neuregulina-1/fisiologia , Potenciais de Ação , Animais , Axônios/metabolismo , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/fisiopatologia , Modelos Animais de Doenças , Técnicas de Introdução de Genes/métodos , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Camundongos Transgênicos , Proteínas Quinases Ativadas por Mitógeno/genética , Neuregulina-1/metabolismo , Neuroglia/metabolismo , Neurônios/metabolismo , Nervos Periféricos/metabolismo , Células de Schwann/metabolismo , Transdução de Sinais/fisiologia
17.
Curr Opin Insect Sci ; 28: 66-72, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-30551769

RESUMO

Initially discovered in bacteria, CRISPR-based genome editing endonucleases have proven remarkably amenable for adaptation to insects. To date, these endonucleases have been utilized in a plethora of both model and non-model insects including diverse flies, bees, beetles, butterflies, moths, and grasshoppers, to name a few, thereby revolutionizing functional genomics of insects. In addition to basic genome editing, they have also been invaluable for advanced genome engineering and synthetic biology applications. Here we explore the recent genome editing advancements in insects for generating site-specific genomic mutations, insertions, deletions, as well as more advanced applications such as Homology Assisted Genome Knock-in (HACK), potential to utilize DNA base editing, generating predictable reciprocal chromosomal translocations, and development gene drives to control the fate of wild populations.


Assuntos
Edição de Genes/métodos , Genoma , Insetos/genética , Animais , Deleção de Genes , Tecnologia de Impulso Genético/métodos , Edição de Genes/instrumentação , Técnicas de Introdução de Genes/métodos , Mutagênese Insercional , Mutação , Translocação Genética
18.
BMC Biol ; 16(1): 151, 2018 12 28.
Artigo em Inglês | MEDLINE | ID: mdl-30593266

RESUMO

BACKGROUND: Cultured human cells are pivotal models to study human gene functions, but introducing complete loss of function in diploid or aneuploid cells has been a challenge. The recently developed CRISPR/Cas9-mediated homology-independent knock-in approach permits targeted insertion of large DNA at high efficiency, providing a tool for insertional disruption of a selected gene. Pioneer studies have showed promising results, but the current methodology is still suboptimal and functional outcomes have not been well examined. Taking advantage of the promoterless fluorescence reporter systems established in our previous study, here, we further investigated potentials of this new insertional gene disruption approach and examined its functional outcomes. RESULTS: Exemplified by using hyperploid LO2 cells, we demonstrated that simultaneous knock-in of dual fluorescence reporters through CRISPR/Cas9-induced homology-independent DNA repair permitted one-step generation of cells carrying complete disruption of target genes at multiple alleles. Through knocking-in at coding exons, we generated stable single-cell clones carrying complete disruption of ULK1 gene at all four alleles, lacking intact FAT10 in all three alleles, or devoid of intact CtIP at both alleles. We have confirmed the depletion of ULK1 and FAT10 transcripts as well as corresponding proteins in the obtained cell clones. Moreover, consistent with previous reports, we observed impaired mitophagy in ULK1-/- cells and attenuated cytokine-induced cell death in FAT10-/- clones. However, our analysis showed that single-cell clones carrying complete disruption of CtIP gene at both alleles preserved in-frame aberrant CtIP transcripts and produced proteins. Strikingly, the CtIP-disrupted clones raised through another two distinct targeting strategies also produced varied but in-frame aberrant CtIP transcripts. Sequencing analysis suggested that diverse DNA processing and alternative RNA splicing were involved in generating these in-frame aberrant CtIP transcripts, and some infrequent events were biasedly enriched among the CtIP-disrupted cell clones. CONCLUSION: Multiallelic gene disruption could be readily introduced through CRISPR/Cas9-induced homology-independent knock-in of dual fluorescence reporters followed by direct tracing and cell isolation. Robust cellular mechanisms exist to spare essential genes from loss-of-function modifications, by generating partially functional transcripts through diverse DNA and RNA processing mechanisms.


Assuntos
Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Sistemas CRISPR-Cas , Proteínas de Transporte/genética , Reparo do DNA , Técnicas de Introdução de Genes/métodos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Nucleares/genética , Ubiquitinas/genética , Linhagem Celular , Mutagênese Insercional
19.
Proc Natl Acad Sci U S A ; 115(36): E8547-E8556, 2018 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-30127005

RESUMO

Inherited retinal degenerations are caused by mutations in >250 genes that affect photoreceptor cells or the retinal pigment epithelium and result in vision loss. For autosomal recessive and X-linked retinal degenerations, significant progress has been achieved in the field of gene therapy as evidenced by the growing number of clinical trials and the recent commercialization of the first gene therapy for a form of congenital blindness. However, despite significant efforts to develop a treatment for the most common form of autosomal dominant retinitis pigmentosa (adRP) caused by >150 mutations in the rhodopsin (RHO) gene, translation to the clinic has stalled. Here, we identified a highly efficient shRNA that targets human (and canine) RHO in a mutation-independent manner. In a single adeno-associated viral (AAV) vector we combined this shRNA with a human RHO replacement cDNA made resistant to RNA interference and tested this construct in a naturally occurring canine model of RHO-adRP. Subretinal vector injections led to nearly complete suppression of endogenous canine RHO RNA, while the human RHO replacement cDNA resulted in up to 30% of normal RHO protein levels. Noninvasive retinal imaging showed photoreceptors in treated areas were completely protected from retinal degeneration. Histopathology confirmed retention of normal photoreceptor structure and RHO expression in rod outer segments. Long-term (>8 mo) follow-up by retinal imaging and electroretinography indicated stable structural and functional preservation. The efficacy of this gene therapy in a clinically relevant large-animal model paves the way for treating patients with RHO-adRP.


Assuntos
Dependovirus , Técnicas de Introdução de Genes/métodos , Técnicas de Silenciamento de Genes/métodos , Terapia Genética/métodos , Vetores Genéticos , RNA Catalítico , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Retinite Pigmentosa , Rodopsina , Animais , Cães , Células HEK293 , Humanos , RNA Catalítico/biossíntese , RNA Catalítico/genética , Células Fotorreceptoras Retinianas Bastonetes/patologia , Retinite Pigmentosa/genética , Retinite Pigmentosa/metabolismo , Retinite Pigmentosa/patologia , Rodopsina/biossíntese , Rodopsina/genética
20.
PLoS One ; 13(5): e0196809, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29847546

RESUMO

Dictyostelium has a mature technology for molecular-genetic manipulation based around transfection using several different selectable markers, marker re-cycling, homologous recombination and insertional mutagenesis, all supported by a well-annotated genome. However this technology is optimized for mutant, axenic cells that, unlike non-axenic wild type, can grow in liquid medium. There is a pressing need for methods to manipulate wild type cells and ones with defects in macropinocytosis, neither of which can grow in liquid media. Here we present a panel of molecular genetic techniques based on the selection of Dictyostelium transfectants by growth on bacteria rather than liquid media. As well as extending the range of strains that can be manipulated, these techniques are faster than conventional methods, often giving usable numbers of transfected cells within a few days. The methods and plasmids described here allow efficient transfection with extrachromosomal vectors, as well as chromosomal integration at a 'safe haven' for relatively uniform cell-to-cell expression, efficient gene knock-in and knock-out and an inducible expression system. We have thus created a complete new system for the genetic manipulation of Dictyostelium cells that no longer requires cell feeding on liquid media.


Assuntos
Dictyostelium/genética , Técnicas de Introdução de Genes/métodos , Engenharia Genética/métodos , Vetores Genéticos/genética , Recombinação Homóloga/genética , Mutagênese Insercional/genética , Mutação/genética , Pinocitose/genética , Plasmídeos/genética , Transfecção/métodos
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