RESUMEN
Sea urchins and other echinoderms are important experimental models for studying developmental processes. The lack of approaches for conditional gene perturbation, however, has made it challenging to investigate the late developmental functions of genes that have essential roles during early embryogenesis and genes that have diverse functions in multiple tissues. The doxycycline-controlled Tet-On system is a widely used molecular tool for temporally and spatially regulated transgene expression. Here, we optimized the Tet-On system to conditionally induce gene expression in sea urchin embryos. Using this approach, we explored the roles the MAPK signaling plays in skeletogenesis by expressing genes that perturb the pathway specifically in primary mesenchyme cells during later stages of development. We demonstrated the wide utility of the Tet-On system by applying it to a second sea urchin species and in cell types other than the primary mesenchyme cells. Our work provides a robust and flexible platform for the spatiotemporal regulation of gene expression in sea urchins, which will considerably enhance the utility of this prominent model system.
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Desarrollo Embrionario , Erizos de Mar , Animales , Erizos de Mar/genética , Expresión Génica , Regulación del Desarrollo de la Expresión GénicaRESUMEN
Pathogenic species within the Rickettsia genus are transmitted to humans through arthropod vectors and cause a spectrum of diseases ranging from mild to life-threatening. Despite rickettsiae posing an emerging global health risk, the genetic requirements of their infectious life cycles remain poorly understood. A major hurdle toward building this understanding has been the lack of efficient tools for genetic manipulation, owing to the technical difficulties associated with their obligate intracellular nature. To this end, we implemented the Tet-On system to enable conditional gene expression in Rickettsia parkeri. Using Tet-On, we show inducible expression of antibiotic resistance and a fluorescent reporter. We further used this inducible promoter to screen the ability of R. parkeri to express four variants of the catalytically dead Cas9 (dCas9). We demonstrate that all four dCas9 variants can be expressed in R. parkeri and used for CRISPR interference (CRISPRi)-mediated targeted gene knockdown. We show targeted knockdown of an antibiotic resistance gene as well as the endogenous virulence factor sca2. Altogether, we have developed systems for inducible gene expression and CRISPRi-mediated gene knockdown for the first time in rickettsiae, laying the groundwork for more scalable, targeted mechanistic investigations into their infectious life cycles.IMPORTANCEThe spotted fever group of Rickettsia contains vector-borne pathogenic bacteria that are neglected and emerging threats to public health. Due to the obligate intracellular nature of rickettsiae, the development of tools for genetic manipulation has been stunted, and the molecular and genetic underpinnings of their infectious lifecycle remain poorly understood. Here, we expand the genetic toolkit by introducing systems for conditional gene expression and CRISPR interference (CRISPRi)-mediated gene knockdown. These systems allow for relatively easy manipulation of rickettsial gene expression. We demonstrate the effectiveness of these tools by disrupting the intracellular life cycle using CRISPRi to deplete the sca2 virulence factor. These tools will be crucial for building a more comprehensive and detailed understanding of rickettsial biology and pathogenesis.
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Sistemas CRISPR-Cas , Regulación Bacteriana de la Expresión Génica , Silenciador del Gen , Rickettsia , Rickettsia/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Regiones Promotoras Genéticas , HumanosRESUMEN
The microRNA-200 (miR-200) family is a potent suppressor of epithelial-to-mesenchymal transition (EMT). While its role as a tumor suppressor has been well documented, recent studies suggested that it can promote cancer progression in several stages. In this study, we investigated whether the miR-200 family members play a role in the acquisition of a hybrid epithelial/mesenchymal (E/M) state, which is reported to be associated with cancer malignancy, in mesenchymal MDA-MB-231 cells. Our results demonstrated that the induction of miR-200c-141, a cluster of the miR-200 family member, can induce the expression of epithelial gene and cell-cell junction while mesenchymal markers are retained. Moreover, induction of miR-200c-141 promoted collective migration accompanied by the formation of F-actin cables anchored by adherens junction. These results suggest that the miR-200 family can induce a hybrid E/M state and endows with the ability of collective cell migration in mesenchymal cancer cells.
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Células MDA-MB-231 , MicroARNs , Línea Celular Tumoral , MicroARNs/genética , MicroARNs/metabolismo , Genes Supresores de Tumor , Movimiento Celular/genética , Transición Epitelial-Mesenquimal/genética , Regulación Neoplásica de la Expresión GénicaRESUMEN
The cone-rod homeobox (CRX) protein is a key transcription factor essential for photoreceptor function and survival. Mutations in human CRX gene are linked to a wide spectrum of blinding diseases ranging from mild macular dystrophy to severe Leber congenital amaurosis (LCA), cone-rod dystrophy (CRD), and retinitis pigmentosa (RP). These diseases are still incurable and mostly inherited in an autosomal dominant form. Dysfunctional mutant CRX protein interferes with the function of wild-type CRX protein, demonstrating the dominant negative effect. At present, gene augmentation is the most promising treatment strategy for hereditary diseases. This study aims to review the pathogenic mechanisms of various CRX mutations and propose two therapeutic strategies to rescue sick photoreceptors in CRX-associated retinopathies, namely, Tet-On-hCRX system and adeno-associated virus (AAV)-mediated gene augmentation. The outcome of proposed studies will guide future translational research and suggest guidelines for therapy evaluation in terms of treatment safety and efficacy.
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Amaurosis Congénita de Leber , Enfermedades de la Retina , Retinitis Pigmentosa , Humanos , Amaurosis Congénita de Leber/genética , Amaurosis Congénita de Leber/terapia , Amaurosis Congénita de Leber/patología , Mutación , Células Fotorreceptoras/patología , Enfermedades de la Retina/genética , Enfermedades de la Retina/terapia , Retinitis Pigmentosa/genética , Retinitis Pigmentosa/terapiaRESUMEN
Immortalization (genetically induced prevention of replicative senescence) is a promising approach to obtain cellular material for cell therapy or for bio-artificial organs aimed at overcoming the problem of donor material shortage. Immortalization is reversed before cells are used in vivo to allow cell differentiation into the mature phenotype and avoid tumorigenic effects of unlimited cell proliferation. However, there is no certainty that the process of de-immortalization is 100% effective and that it does not cause unwanted changes in the cell. In this review, we discuss various approaches to reversible immortalization, emphasizing their advantages and disadvantages in terms of biosafety. We describe the most promising approaches in improving the biosafety of reversibly immortalized cells: CRISPR/Cas9-mediated immortogene insertion, tamoxifen-mediated self-recombination, tools for selection of successfully immortalized cells, using a decellularized extracellular matrix, and ensuring post-transplant safety with the use of suicide genes. The last process may be used as an add-on for previously existing reversible immortalized cell lines.
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Contención de Riesgos Biológicos , Telomerasa , Línea Celular , Diferenciación Celular , Proliferación Celular , Telomerasa/metabolismoRESUMEN
Filamentous fungal secondary metabolites are an important source of bioactive components. Genome sequencing ofAspergillus terreusrevealed many silent secondary metabolite biosynthetic gene clusters presumed to be involved in producing secondary metabolites. Activation of silent gene clusters through overexpressing a pathway-specific regulator is an effective avenue for discovering novel fungal secondary metabolites. Replacement of the native promoter of the pathway-specific activator with the inducible Tet-on system to activate thetazpathway led to the discovery of a series of azaphilone secondary metabolites, among which azaterrilone A (1) was purified and identified for the first time. Genetic deletion of core PKS genes and transcriptional analysis further characterized thetazgene cluster to consist of 16 genes with the NR-PKS and the HR-PKS collaborating in a convergent mode. Based on the putative gene functions and the characterized compounds structural information, a biosynthetic pathway of azaterrilone A (1) was proposed.
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Aspergillus , Familia de Multigenes , Aspergillus/genética , Aspergillus/metabolismo , Benzopiranos , Pigmentos Biológicos/genética , Pigmentos Biológicos/metabolismo , Sintasas Poliquetidas/genética , Sintasas Poliquetidas/metabolismoRESUMEN
BACKGROUND: Our recent multi-omics analyses of glucoamylase biosynthesis in Aspergillus niger (A. niger) suggested that lipid catabolism was significantly up-regulated during high-yield period under oxygen limitation. Since the catabolism of fatty acids can provide energy compounds such as ATP and important precursors such as acetyl-CoA, we speculated that enhancement of this pathway might be beneficial to glucoamylase overproduction. RESULTS: Based on previous transcriptome data, we selected and individually overexpressed five candidate genes involved in fatty acid degradation under the control of the Tet-on gene switch in A. niger. Overexpression of the fadE, fadA and cyp genes increased the final specific enzyme activity and total secreted protein on shake flask by 21.3 ~ 31.3% and 16.0 ~ 24.2%, respectively. And a better inducible effect by doxycycline was obtained from early logarithmic growth phase (18 h) than stationary phase (42 h). Similar with flask-level results, the glucoamylase content and total extracellular protein in engineered strains OE-fadE (overexpressing fadE) and OE-fadA (overexpressing fadA) on maltose-limited chemostat cultivation were improved by 31.2 ~ 34.1% and 35.1 ~ 38.8% compared to parental strain B36. Meanwhile, intracellular free fatty acids were correspondingly decreased by 41.6 ~ 44.6%. The metabolomic analysis demonstrated intracellular amino acids pools increased 24.86% and 18.49% in two engineered strains OE-fadE and OE-fadA compared to B36. Flux simulation revealed that increased ATP, acetyl-CoA and NADH was supplied into TCA cycle to improve amino acids synthesis for glucoamylase overproduction. CONCLUSION: This study suggested for the first time that glucoamylase production was significantly improved in A. niger by overexpression of genes fadE and fadA involved in fatty acids degradation pathway. Harnessing the intracellular fatty acids could be a strategy to improve enzyme production in Aspergillus niger cell factory.
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Aspergillus niger , Glucano 1,4-alfa-Glucosidasa , Glucano 1,4-alfa-Glucosidasa/metabolismo , Aspergillus niger/metabolismo , Acetilcoenzima A/metabolismo , Aminoácidos/metabolismo , Ácidos Grasos/metabolismo , Adenosina Trifosfato/metabolismoRESUMEN
Galectin-4 (Gal4) has been suggested to function as a tumor suppressor in colorectal cancer (CRC). In order to systematically explore its function in CRC, we established a CRC cell line where Gal4 expression can be regulated via the doxycycline (dox)-inducible expression of a single copy wildtype LGALS4 transgene generated by recombinase-mediated cassette exchange (RMCE). Using this model and applying in-depth proteomic and phosphoproteomic analyses, we systematically screened for intracellular changes induced by Gal4 expression. Overall, 3083 cellular proteins and 2071 phosphosites were identified and quantified, of which 1603 could be matched and normalized to their protein expression levels. A bioinformatic analysis revealed that most of the regulated proteins and phosphosites can be localized in the nucleus and are categorized as nucleic acid-binding proteins. The top candidates whose expression was modulated by Gal4 are PURB, MAPKAPK3, BTF3 and BCAR1, while the prime candidates with altered phosphorylation included ZBTB7A, FOXK1, PURB and CK2beta. In order to validate the (phospho)proteomic data, we confirmed these candidates by a radiometric metabolic-labelling and immunoprecipitation strategy. All candidates exert functions in the transcriptional or translational control, indicating that Gal4 might be involved in these processes by affecting the expression or activity of these proteins.
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Neoplasias Colorrectales , Proteómica , Línea Celular Tumoral , Neoplasias Colorrectales/patología , Proteínas de Unión al ADN , Factores de Transcripción Forkhead , Galectina 4 , Humanos , Espacio Intracelular/metabolismo , Proteómica/métodos , Recombinasas , Factores de TranscripciónRESUMEN
Precise analysis of the genetic expression and functioning of proteins requires experimental approaches that, among others, enable tight control of gene expression at the transcriptional level. Doxycycline-induced Tet-On/Tet-Off expression systems provide such an opportunity, and are frequently used to regulate the activity of genes in eukaryotic cells. Since its development, the Tet-system has evolved tight gene control in mammalian cells; however, some challenges are still unaddressed. In the current set up, the establishment of the standard Tet-based system in target cells is time-consuming and laborious and has been shown to be inefficient, especially in a long-term perspective. In this work, we present an optimized inducible expression system, which enables rapid generation of doxycycline-responsive cells according to a one- or two-step protocol. The reported modifications of the Tet-On system expand the toolbox for regulated mammalian gene expression and provide high, stable, and homogenous expression of the Tet-On3G transactivator, which is of fundamental importance in the regulation of transgenes.
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Antibacterianos/farmacología , Regulación de la Expresión Génica , Técnicas Genéticas , Vectores Genéticos/genética , Animales , Doxiciclina/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas Fluorescentes Verdes/genética , Células HEK293 , Células HeLa , Humanos , Ratones , Células 3T3 NIH , Proteína Ribosómica L10/genética , Tetraciclina/farmacología , Transactivadores/genética , TransgenesRESUMEN
BMP-7 has shown inductive potential for in vitro osteogenic differentiation of mesenchymal stem cells, which are an ideal resource for regenerative medicine. Externally applied, recombinant BMP-7 was able to induce the osteogenic differentiation of DPSCs but based on our previous results with BMP-2, we aimed to study the effect of the tetracyclin-inducible BMP-7 expression on these cells. DPSC, mock, and DPSC-BMP-7 cell lines were cultured in the presence or absence of doxycycline, then alkaline phosphatase (ALP) activity, mineralization, and mRNA levels of different osteogenic marker genes were measured. In the DPSC-BMP-7 cell line, the level of BMP-7 mRNA significantly increased in the media supplemented with doxycycline, however, the expression of Runx2 and noggin genes was upregulated only after 21 days of incubation in the osteogenic medium with doxycycline. Moreover, while the examination of ALP activity showed reduced activity in the control medium containing doxycycline, the accumulation of minerals remained unchanged in the cultures. We have found that the induced BMP-7 expression failed to induce osteogenic differentiation of DPSCs. We propose three different mechanisms that may worth investigating for the engineering of expression systems that can be used for the induction of differentiation of mesenchymal stem cells.
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Proteína Morfogenética Ósea 7/metabolismo , Diferenciación Celular , Pulpa Dental/citología , Doxiciclina/farmacología , Osteogénesis , Células Madre/citología , Fosfatasa Alcalina/metabolismo , Antibacterianos/farmacología , Proliferación Celular , Células Cultivadas , Pulpa Dental/efectos de los fármacos , Pulpa Dental/metabolismo , Humanos , Células Madre/efectos de los fármacos , Células Madre/metabolismoRESUMEN
To determine how nuclease deactivated Cas9 (dCas9) or single-guide RNA (sgRNA) expression levels affect the knockdown efficiency of CRISPRi, we created K562 cell clones expressing KRAB-dCas9 protein either with the inducible Tet-on system or with the constitutive SFFV promotor. Single clones were selected by fluorescence-activated cell sorting (FACS) for further study. Six genes with various expression levels were targeted using lentiviral sgRNA from two libraries in four cell clones with various KRAB-dCas9 expression levels. The expression level of dCas9 protein/sgRNA levels and the knockdown efficiency were determined by flow cytometry. The cell clone with the highest KRAB-dCas9 expression level achieved effective CRISPRi knockdown. The data describing this clone were statistically different from that on other clones, indicating the strong KRAB-dCas9 expression might be a prerequisite for CRISPRi. By adopting different multiplicity of infection (MOI) in lentiviral transduction of this clone, we modified the expression level of sgRNA and found that the knockdown efficiency was neither affected by the target gene expression level nor correlated with KRAB-dCas9 levels, which remained relatively constant across all knockdown experiments (coefficient of variation = 2.2%). As an example, the following levels of the knockdowns: 74.72, 72.28 and 39.08% for mmadhc, rpia and znf148 genes, respectively, were achieved. These knockdown efficiencies correlated well with the respective sgRNA expression levels. Linear regression models built using this data indicate that the knockdown efficiency may be significantly affected by the levels of both KRAB-dCas9 and sgRNA. Notably, the sgRNA levels have greater impact, being a major factor affecting CRISPRi efficiency.
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Sistemas CRISPR-Cas , ARN Guía de Kinetoplastida , Proteína 9 Asociada a CRISPR , Sistemas CRISPR-Cas/genética , Proteínas de Unión al ADN , Humanos , Células K562 , Regiones Promotoras Genéticas , Factores de TranscripciónRESUMEN
Genetic code expansion (GCE) is a versatile tool to site-specifically incorporate a noncanonical amino acid (ncAA) into a protein, for example, to perform fluorescent labeling inside living cells. To this end, an orthogonal aminoacyl-tRNA-synthetase/tRNA (RS/tRNA) pair is used to insert the ncAA in response to an amber stop codon in the protein of interest. One of the drawbacks of this system is that, in order to achieve maximum efficiency, high levels of the orthogonal tRNA are required, and this could interfere with host cell functionality. To minimize the adverse effects on the host, we have developed an inducible GCE system that enables us to switch on tRNA or RS expression when needed. In particular, we tested different promotors in the context of the T-REx or Tet-On systems to control expression of the desired orthogonal tRNA and/or RS. We discuss our result with respect to the control of GCE components as well as efficiency. We found that only the T-REx system enables simultaneous control of tRNA and RS expression.
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Aminoácidos/genética , Aminoacil-ARNt Sintetasas/genética , Eucariontes/genética , ARN de Transferencia/genética , Aminoácidos/metabolismo , Aminoacil-ARNt Sintetasas/metabolismo , Escherichia coli/citología , Escherichia coli/genética , Escherichia coli/metabolismo , Eucariontes/metabolismo , Código Genético , Células HEK293 , Humanos , ARN de Transferencia/metabolismoRESUMEN
BACKGROUND: Aspergillus niger is a filamentous fungus used for the majority of global citric acid production. Recent developments in genome editing now enable biotechnologists to engineer and optimize A. niger. Currently, however, genetic-leads for maximizing citric acid titers in industrial A. niger isolates is limited. RESULTS: In this study, we try to engineer two citric acid A. niger production isolates, WT-D and D353, to serve as platform strains for future high-throughput genome engineering. Consequently, we used genome editing to simultaneously disrupt genes encoding the orotidine-5'-decarboxylase (pyrG) and non-homologous end-joining component (kusA) to enable use of the pyrG selection/counter selection system, and to elevate homologous recombination rates, respectively. During routine screening of these pyrG mutant strains, we unexpectedly observed a 2.17-fold increase in citric acid production when compared to the progenitor controls, indicating that inhibition of uridine/pyrimidine synthesis may increase citric acid titers. In order to further test this hypothesis, the pyrG gene was placed under the control of a tetracycline titratable cassette, which confirmed that reduced expression of this gene elevated citric acid titers in both shake flask and bioreactor fermentation. Subsequently, we conducted intracellular metabolomics analysis, which demonstrated that pyrG disruption enhanced the glycolysis flux and significantly improved abundance of citrate and its precursors. CONCLUSIONS: In this study, we deliver two citric acid producing isolates which are amenable to high throughput genetic manipulation due to pyrG/kusA deletion. Strikingly, we demonstrate for the first time that A. niger pyrG is a promising genetic lead for generating citric acid hyper-producing strains. Our data support the hypothesis that uridine/pyrimidine biosynthetic pathway offer future avenues for strain engineering efforts.
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Aspergillus niger/genética , Ácido Cítrico/metabolismo , Edición Génica/métodos , Uridina/análogos & derivados , Uridina/metabolismoRESUMEN
BACKGROUND: Nicotinamide adenine dinucleotide phosphate (NADPH) is an important cofactor ensuring intracellular redox balance, anabolism and cell growth in all living systems. Our recent multi-omics analyses of glucoamylase (GlaA) biosynthesis in the filamentous fungal cell factory Aspergillus niger indicated that low availability of NADPH might be a limiting factor for GlaA overproduction. RESULTS: We thus employed the Design-Build-Test-Learn cycle for metabolic engineering to identify and prioritize effective cofactor engineering strategies for GlaA overproduction. Based on available metabolomics and 13C metabolic flux analysis data, we individually overexpressed seven predicted genes encoding NADPH generation enzymes under the control of the Tet-on gene switch in two A. niger recipient strains, one carrying a single and one carrying seven glaA gene copies, respectively, to test their individual effects on GlaA and total protein overproduction. Both strains were selected to understand if a strong pull towards glaA biosynthesis (seven gene copies) mandates a higher NADPH supply compared to the native condition (one gene copy). Detailed analysis of all 14 strains cultivated in shake flask cultures uncovered that overexpression of the gsdA gene (glucose 6-phosphate dehydrogenase), gndA gene (6-phosphogluconate dehydrogenase) and maeA gene (NADP-dependent malic enzyme) supported GlaA production on a subtle (10%) but significant level in the background strain carrying seven glaA gene copies. We thus performed maltose-limited chemostat cultures combining metabolome analysis for these three isolates to characterize metabolic-level fluctuations caused by cofactor engineering. In these cultures, overexpression of either the gndA or maeA gene increased the intracellular NADPH pool by 45% and 66%, and the yield of GlaA by 65% and 30%, respectively. In contrast, overexpression of the gsdA gene had a negative effect on both total protein and glucoamylase production. CONCLUSIONS: This data suggests for the first time that increased NADPH availability can indeed underpin protein and especially GlaA production in strains where a strong pull towards GlaA biosynthesis exists. This data also indicates that the highest impact on GlaA production can be engineered on a genetic level by increasing the flux through the pentose phosphate pathway (gndA gene) followed by engineering the flux through the reverse TCA cycle (maeA gene). We thus propose that NADPH cofactor engineering is indeed a valid strategy for metabolic engineering of A. niger to improve GlaA production, a strategy which is certainly also applicable to the rational design of other microbial cell factories.
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Aspergillus niger/genética , Aspergillus niger/metabolismo , Coenzimas/metabolismo , Glucano 1,4-alfa-Glucosidasa/biosíntesis , Ingeniería Metabólica , Biosíntesis de Proteínas , Coenzimas/genética , NADP/metabolismo , Vía de Pentosa FosfatoRESUMEN
BACKGROUND: To obtain a deep understanding of the mechanism by which breast cancer develops, the genes involved in tumorigenesis should be analyzed in vivo. Mouse mammary gland can regenerate completely from a mammary stem cell (MaSC), which enables us to analyze the effect of gene expression and repression on tumorigenesis in mammary gland regenerated from genetically manipulated MaSCs. Although lentiviral and retroviral systems have usually been applied for gene transduction into MaSCs, they are associated with difficulty in introducing long, repeated, or transcriptional termination sequences. There is thus a need for an easier and quicker gene delivery system. METHODS: We devised a new system for gene delivery into MaSCs using the piggyBac transposon vectors and electroporation. Compared with viral systems, this system enables easier and quicker transfection of even long, repeated, or transcriptional termination DNA sequences. We designed gene expression vectors of the transposon system, equipped with a luciferase (Luc) expression cassette for monitoring gene transduction into regenerative mammary gland in mice by in-vivo imaging. A doxycycline (Dox)-inducible system was also integrated for expressing the target gene after mammary regeneration to mimic the actual mechanism of tumorigenesis. RESULTS: With this new gene delivery system, genetically manipulated mammary glands were successfully reconstituted even though the vector size was > 200 kb and even in the presence of DNA elements such as promoters and transcription termination sequences, which are major obstacles to viral vector packaging. They differentiated correctly into both basal and luminal cells, and showed normal morphological change and milk production after pregnancy, as well as self-renewal capacity. Using the Tet-On system, gene expression can be controlled by the addition of Dox after mammary reconstitution. In a case study using polyoma-virus middle T antigen (PyMT), oncogene-induced tumorigenesis was achieved. The histological appearance of the tumor was highly similar to that of the mouse mammary tumor virus-PyMT transgenic mouse model. CONCLUSIONS: With this system, gene transduction in the mammary gland can be easily and quickly achieved, and gene expression can be controlled by Dox administration. This system for genetic manipulation could be useful for analyzing genes involved in breast cancer.
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Diferenciación Celular/genética , Ingeniería Genética/métodos , Glándulas Mamarias Animales/fisiología , Neoplasias Mamarias Experimentales/genética , Células Madre/fisiología , Animales , Línea Celular , Elementos Transponibles de ADN/genética , Proteínas de Unión al ADN/genética , Doxiciclina/administración & dosificación , Femenino , Fibroblastos , Regulación de la Expresión Génica/efectos de los fármacos , Genes Reporteros , Vectores Genéticos/genética , Proteínas Fluorescentes Verdes/genética , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/trasplante , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Cultivo Primario de Células/métodos , Transfección/métodosRESUMEN
Eukaryotic inducible overexpression systems, including Tet-On and mifepristone-inducible systems, have been widely used to study gene functions by reverse genetics. Among the transposon systems reported to date, the piggyBac transposon system is one of the most efficient in cultured mammalian cells. Here, we report a piggyBac-based double-inducible system that combined the advantages of previous systems. To create this system, the trans- and cis-elements of the Tet-On and mifepristone-inducible systems were cloned into a piggyBac-based trans-vector and cis-vector, respectively. The coding regions of two splicing variants of RUNX1, RUNX1a and RUNX1b, were inserted into the cis-vector to test its ability to express foreign genes along with fluorescent marker proteins. Transgenic 293â¯T cells were established, and the system was tested by inducing expression of foreign genes with DOX and/or mifepristone; GFP and/or mCherry were used as reporter genes. The system efficiently and stringently induced expression of GFP/mCherry and their co-expressed genes without significant mutual interference, as determined by qRT-PCR and Western blot. This piggyBac-based double-inducible system represents a new genetic tool for studying gene functions and interactions in vitro and in vivo in almost all organisms.
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Elementos Transponibles de ADN/genética , Vectores Genéticos/genética , Plásmidos/genética , Clonación Molecular , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Doxiciclina/farmacología , Regulación de la Expresión Génica/genética , Genes Reporteros/genética , Proteínas Fluorescentes Verdes/genética , Células HEK293 , Humanos , Secuencias Reguladoras de Ácidos Nucleicos/genéticaRESUMEN
The filamentous fungus Aspergillus niger is widely used in the biotechnology industry for the production of chemicals and enzymes. Engineering of this valuable organism to improve its productivity is currently hampered by the lack of efficient genetic tools. Here, a Cre-loxP-based system for gene editing in A. niger was developed and its application in construction of A. niger cell factories to produce various organic acids was explored. Two established inducible systems, the xylanase A gene promoter Pxln and Tet-on system, were examined for driving cre expression and thus selection marker hyh deletion. Under inducing conditions, the efficiency of loxP site-specific recombination in the strain with cre driven by Pxln is about 2%, while cre driven by Tet-on system is about 34% which was used as the platform strain for further genetic engineering. As a proof of application of this system, strains containing different copies of oxaloacetate acetylhydrolase-encoding gene (oahA) were constructed, and the resultant strain S428 showed as high as 3.1-fold increase in oxalic acid production. Furthermore, an efficient malate-producing strain was generated through four-step genetic manipulation (oahA deletion, pyc, mdh3 and C4-dicarboxylate transporter gene c4t318 insertion). The resultant strain S575 achieved a titer 120.38 g/L malic acid with the flask culture, and a titer 201.24 g/L malic acid in fed-batch fermentation. These results demonstrated that this modified Cre-loxP system is a powerful tool for genetic engineering in A. niger, which has the potential to be genetically modified as a viable aciduric platform strain to produce high levels of various organic acids.
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Aspergillus niger/genética , Aspergillus niger/metabolismo , Ácidos Carboxílicos/metabolismo , Edición Génica/métodos , Ingeniería Metabólica/métodos , Redes y Vías Metabólicas/genética , Recombinación GenéticaRESUMEN
The adult human adipose tissue is predominantly composed of white adipocytes. However, within certain depots, adipose tissue contains thermogenically active brown-like adipocytes, which have been evolutionarily conserved in mammals. This chapter will give a brief overview on the methods used to genetically target and trace both white and brown adipocytes using techniques such as bacterial artificial chromosome (BAC) cloning to create transgenic mouse models and the tools with which genetic recombination is mediated in vivo (e.g., Cre-loxP, CreERT, and Tet-On). The chapter furthermore critically discusses the strength and limitation of the various systems used to target mature white and brown adipocytes (ap2-Cre, Adipoq-Cre, and Ucp1-Cre). Based on these systems, it is evident that our knowledge of mature adipocyte categorization into brown, white, brite, or beige adipocytes is strongly influenced by the use of the various genetic mouse models described in this chapter. Our evaluation of different studies using the aforementioned systems focuses on key genes, which have been reported to maintain adipocyte's function (insulin receptor, Raptor, or Atgl).
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Adipocitos Marrones , Adipocitos Blancos/metabolismo , Tejido Adiposo Pardo/metabolismo , Tejido Adiposo/fisiología , Integrasas , Adipocitos Blancos/fisiología , Tejido Adiposo Pardo/fisiología , Adulto , Animales , Humanos , Ratones , Ratones TransgénicosRESUMEN
Antigen-presenting cells (APCs) including dendritic cells (DCs) play a critical role in the development of autoimmune diseases by presenting self-antigen to T-cells. Different signals modulate the ability of APCs to activate or tolerize autoreactive T-cells. Since the expression of heme oxygenase-1 (HO-1) by APCs has been associated with the tolerization of autoreactive T-cells, we hypothesized that HO-1 expression might be altered in APCs from autoimmune-prone non-obese diabetic (NOD) mice. We found that, compared to control mice, NOD mice exhibited a lower percentage of HO-1-expressing cells among the splenic DCs, suggesting an impairment of their tolerogenic functions. To investigate whether restored expression of HO-1 in APCs could alter the development of diabetes in NOD mice, we generated a transgenic mouse strain in which HO-1 expression can be specifically induced in DCs using a tetracycline-controlled transcriptional activation system. Mice in which HO-1 expression was induced in DCs exhibited a lower Type 1 Diabetes (T1D) incidence and a reduced insulitis compared to non-induced mice. Upregulation of HO-1 in DCs also prevented further increase of glycemia in recently diabetic NOD mice. Altogether, our data demonstrated the potential of induction of HO-1 expression in DCs as a preventative treatment, and potential as a curative approach for T1D.
Asunto(s)
Diabetes Mellitus Tipo 1/enzimología , Diabetes Mellitus Tipo 1/prevención & control , Hemo-Oxigenasa 1/genética , Animales , Antígeno CD11c/metabolismo , Células Dendríticas/metabolismo , Diabetes Mellitus Tipo 1/complicaciones , Doxiciclina/farmacología , Hiperglucemia/complicaciones , Hiperglucemia/prevención & control , Ratones Endogámicos NOD , Ratones Transgénicos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
BACKGROUND: The lifestyle of filamentous fungi depends on the secretion of hydrolytic enzymes into the surrounding medium, which degrade polymeric substances into monomers that are then taken up to sustain metabolism. This feature has been exploited in biotechnology to establish platform strains with high secretory capacity including Aspergillus niger. The accepted paradigm is that proteins become mainly secreted at the tips of fungal hyphae. However, it is still a matter of debate if the amount of growing hyphal tips in filamentous fungi correlates with an increase in secretion, with previous studies showing either a positive or no correlation. RESULTS: Here, we followed a systematic approach to study protein secretion in A. niger. First, we put the glaA gene encoding for glucoamylase (GlaA), the most abundant secreted protein of A. niger, under control of the tunable Tet-on system. Regulation of glaA gene expression by omitting or adding the inducer doxycycline to cultivation media allowed us to study the effect of glaA under- or overexpression in the same isolate. By inducing glaA expression in a fluorescently tagged v-SNARE reporter strain expressing GFP-SncA, we could demonstrate that the amount of post-Golgi carriers indeed depends on and correlates with glaA gene expression. By deleting the racA gene, encoding the Rho-GTPase RacA in this isolate, we generated a strain which is identical to the parental strain with respect to biomass formation but produces about 20% more hyphal tips. This hyperbranching phenotype caused a more compact macromorphology in shake flask cultivations. When ensuring continuous high-level expression of glaA by repeated addition of doxycycline, this hyperbranching strain secreted up to four times more GlaA into the culture medium compared to its parental strain. CONCLUSION: The data obtained in this study strongly indicate that A. niger responds to forced transcription of secretory enzymes with increased formation of post-Golgi carriers to efficiently accommodate the incoming cargo load. This physiological adaptation can be rationally exploited to generate hypersecretion platforms based on a hyperbranching phenotype. We propose that a racA deletion background serves as an excellent chassis for such hypersecretion strains.