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1.
Fungal Genet Biol ; 160: 103694, 2022 05.
Artículo en Inglés | MEDLINE | ID: mdl-35398258

RESUMEN

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.


Asunto(s)
Aspergillus , Familia de Multigenes , Aspergillus/genética , Aspergillus/metabolismo , Benzopiranos , Pigmentos Biológicos/genética , Pigmentos Biológicos/metabolismo , Sintasas Poliquetidas/genética , Sintasas Poliquetidas/metabolismo
2.
Int J Mol Sci ; 23(12)2022 Jun 08.
Artículo en Inglés | MEDLINE | ID: mdl-35742860

RESUMEN

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.


Asunto(s)
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 Transcripción
3.
Mol Biol (Mosk) ; 55(1): 86-95, 2021.
Artículo en Ruso | MEDLINE | ID: mdl-33566028

RESUMEN

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.


Asunto(s)
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 Transcripción
4.
Microb Cell Fact ; 19(1): 76, 2020 Mar 24.
Artículo en Inglés | MEDLINE | ID: mdl-32209089

RESUMEN

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.


Asunto(s)
Aspergillus niger/genética , Ácido Cítrico/metabolismo , Edición Génica/métodos , Uridina/análogos & derivados , Uridina/metabolismo
5.
Breast Cancer Res ; 21(1): 1, 2019 01 05.
Artículo en Inglés | MEDLINE | ID: mdl-30611295

RESUMEN

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.


Asunto(s)
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/métodos
6.
Plasmid ; 105: 102420, 2019 09.
Artículo en Inglés | MEDLINE | ID: mdl-31265838

RESUMEN

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 293 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.


Asunto(s)
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/genética
7.
Appl Microbiol Biotechnol ; 103(19): 8105-8114, 2019 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-31392377

RESUMEN

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.


Asunto(s)
Aspergillus niger/genética , Aspergillus niger/metabolismo , Ácidos Carboxílicos/metabolismo , Edición Génica/métodos , Ingeniería Metabólica/métodos , Redes y Vías Metabólicas/genética , Recombinación Genética
8.
Int J Mol Sci ; 20(7)2019 Apr 03.
Artículo en Inglés | MEDLINE | ID: mdl-30987262

RESUMEN

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 fármacos
9.
Microb Cell Fact ; 17(1): 128, 2018 Aug 21.
Artículo en Inglés | MEDLINE | ID: mdl-30129427

RESUMEN

BACKGROUND: Filamentous fungi including Aspergillus niger are cell factories for the production of organic acids, proteins and bioactive compounds. Traditionally, stirred-tank reactors (STRs) are used to cultivate them under highly reproducible conditions ensuring optimum oxygen uptake and high growth rates. However, agitation via mechanical stirring causes high shear forces, thus affecting fungal physiology and macromorphologies. Two-dimensional rocking-motion wave-mixed bioreactor cultivations could offer a viable alternative to fungal cultivations in STRs, as comparable gas mass transfer is generally achievable while deploying lower friction and shear forces. The aim of this study was thus to investigate for the first time the consequences of wave-mixed cultivations on the growth, macromorphology and product formation of A. niger. RESULTS: We investigated the impact of hydrodynamic conditions on A. niger cultivated at a 5 L scale in a disposable two-dimensional rocking motion bioreactor (CELL-tainer®) and a BioFlo STR (New Brunswick®), respectively. Two different A. niger strains were analysed, which produce heterologously the commercial drug enniatin B. Both strains expressed the esyn1 gene that encodes a non-ribosomal peptide synthetase ESYN under control of the inducible Tet-on system, but differed in their dependence on feeding with the precursors D-2-hydroxyvaleric acid and L-valine. Cultivations of A. niger in the CELL-tainer resulted in the formation of large pellets, which were heterogeneous in size (diameter 300-800 µm) and not observed during STR cultivations. When talcum microparticles were added, it was possible to obtain a reduced pellet size and to control pellet heterogeneity (diameter 50-150 µm). No foam formation was observed under wave-mixed cultivation conditions, which made the addition of antifoam agents needless. Overall, enniatin B titres of about 1.5-2.3 g L-1 were achieved in the CELL-tainer® system, which is about 30-50% of the titres achieved under STR conditions. CONCLUSIONS: This is the first report studying the potential use of single-use wave-mixed reactor systems for the cultivation of A. niger. Although final enniatin yields are not competitive yet with titres achieved under STR conditions, wave-mixed cultivations open up new avenues for the cultivation of shear-sensitive mutant strains as well as high cell-density cultivations.


Asunto(s)
Aspergillus niger/genética , Reactores Biológicos
10.
J Gene Med ; 19(1-2)2017 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-28009940

RESUMEN

BACKGROUND: Hydrodynamic tail vein injection (HTVI) of transposon-based integration vectors is an established system for stably transfecting mouse hepatocytes in vivo that has been successfully employed to study key questions in liver biology and cancer. Refining the vectors for transposon-mediated hepatocyte transfection will further expand the range of applications of this technique in liver research. In the present study, we report an advanced transposon-based system for manipulating gene expression in hepatocytes in vivo. METHODS: Transposon-based vector constructs were generated to enable the constitutive expression of inducible Cre recombinase (CreER) together with tetracycline-inducible transgene or miR-small hairpin RNA (shRNA) expression (Tet-ON system). Transposon and transposase expression vectors were co-injected into R26R-mTmG reporter mice by HTVI. Cre-mediated gene recombination was induced by tamoxifen, followed by the administration of doxycycline to drive tetracycline-inducible gene or shRNA expression. Expression was visualized by immunofluorescence staining in livers of injected mice. RESULTS: After HTVI, Cre recombination by tamoxifen led to the expression of membrane-bound green fluorescent protein in transfected hepatocytes. Activation of inducible gene or shRNA expression was detected by immunostaining in up to one-third of transfected hepatocytes, with an efficiency dependent on the promoter driving the Tet-ON system. CONCLUSIONS: Our vector system combines Cre-lox mediated gene mutation with inducible gene expression or gene knockdown, respectively. It provides the opportunity for rapid and specific modification of hepatocyte gene expression and can be a useful tool for genetic screening approaches and analysis of target genes specifically in genetically engineered mouse models.


Asunto(s)
Expresión Génica , Silenciador del Gen , Hepatocitos/metabolismo , Transfección , Animales , Elementos Transponibles de ADN , Doxiciclina/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Técnicas de Inactivación de Genes , Orden Génico , Genes Reporteros , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Recombinación Homóloga , Ratones , Regiones Promotoras Genéticas , ARN Interferente Pequeño/genética , Transfección/métodos , Transgenes
11.
Nanomedicine ; 13(4): 1363-1375, 2017 05.
Artículo en Inglés | MEDLINE | ID: mdl-28219741

RESUMEN

The human glial-cell derived neurotrophic factor (hGDNF) gene transfer by neurotensin (NTS)-polyplex nanoparticles functionally restores the dopamine nigrostriatal system in experimental Parkinson's disease models. However, high levels of sustained expression of GDNF eventually can cause harmful effects. Herein, we report an improved NTS-polyplex nanoparticle system that enables regulation of hGDNF expression within dopaminergic neurons. We constructed NTS-polyplex nanoparticles containing a single bifunctional plasmid that codes for the reverse tetracycline-controlled transactivator advanced (rtTA-Adv) under the control of NBRE3x promoter, and for hGDNF under the control of tetracycline-response element (TRE). Another bifunctional plasmid contained the enhanced green fluorescent protein (GFP) gene. Transient transfection experiments in N1E-115-Nurr1 cells showed that doxycycline (100 ng/mL) activates hGDNF and GFP expression. Doxycycline (5 mg/kg, i.p.) administration in rats activated hGDNF expression only in transfected dopaminergic neurons, whereas doxycycline withdrawal silenced transgene expression. Our results offer a specific doxycycline-regulated system suitable for nanomedicine-based treatment of Parkinson's disease.


Asunto(s)
Neuronas Dopaminérgicas/metabolismo , Doxiciclina/farmacología , Regulación de la Expresión Génica , Nanopartículas/química , Neurotensina/química , Miembro 1 del Grupo A de la Subfamilia 6 de Receptores Nucleares/genética , Animales , Línea Celular Tumoral , Vectores Genéticos , Humanos , Masculino , Ratones , Miembro 1 del Grupo A de la Subfamilia 6 de Receptores Nucleares/metabolismo , Enfermedad de Parkinson/tratamiento farmacológico , Plásmidos , Regiones Promotoras Genéticas , Ratas , Ratas Wistar , Elementos de Respuesta , Transfección , Transgenes
12.
Fungal Genet Biol ; 89: 84-88, 2016 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-26851300

RESUMEN

Genome sequencing of the fungus Aspergillus terreus uncovered a number of silent core structural biosynthetic genes encoding enzymes presumed to be involved in the production of cryptic secondary metabolites. There are five nonribosomal peptide synthetase (NRPS)-like genes with the predicted A-T-TE domain architecture within the A. terreus genome. Among the five genes, only the product of pgnA remains unknown. The Tet-on system is an inducible, tunable and metabolism-independent expression system originally developed for Aspergillus niger. Here we report the adoption of the Tet-on system as an effective gene activation tool in A. terreus. Application of this system in A. terreus allowed us to uncover the product of the cryptic NRPS-like gene, pgnA. Furthermore expression of pgnA in the heterologous Aspergillus nidulans host suggested that the pgnA gene alone is necessary for phenguignardic acid (1) biosynthesis.


Asunto(s)
Aspergillus/enzimología , Aspergillus/genética , Doxiciclina/farmacología , Genes Fúngicos/genética , Péptido Sintasas/genética , Aspergillus/efectos de los fármacos , Aspergillus/metabolismo , Aspergillus nidulans/efectos de los fármacos , Aspergillus nidulans/genética , Aspergillus niger/efectos de los fármacos , Aspergillus niger/genética , Productos Biológicos/metabolismo , Dioxolanos/metabolismo , Familia de Multigenes , Activación Transcripcional
13.
Genesis ; 52(5): 408-16, 2014 May.
Artículo en Inglés | MEDLINE | ID: mdl-24585429

RESUMEN

Sphingomyelin phosphodiesterase 3 (SMPD3) is a pleiotropic lipid metabolizing enzyme involved in multiple physiological processes. A deletion mutation in the murine Smpd3 gene called fragilitas ossium (fro) leads to severe skeletal abnormalities in the developing fro/fro embryos. Although fro/fro mice can be useful to study many different aspects of SMPD3 functions, their perinatal lethality makes it difficult to generate a sufficient number of mice for controlled studies. In fact, on the C57BL/6 genetic background, none of the fro/fro mice survive beyond the perinatal stage. In this study, we used the "Tet-On" inducible gene expression system to express Smpd3 transiently in fro/fro;ROSA-rtTA;TRE-Smpd3 embryos on the C57BL/6 background. This induced Smpd3 expression corrected all the skeletal abnormalities in these embryos and prevented their early death. However, induction of Smpd3 expression in the adolescent fro/fro;ROSA-rtTA;TRE-Smpd3 mice was not sufficient to correct the defects in trabecular bone mineralization and the impaired growth of the long bones. This novel mouse model will be a useful tool to study SMPD3 biology in vivo.


Asunto(s)
Genes Letales , Osteogénesis Imperfecta/embriología , Osteogénesis , Esfingomielina Fosfodiesterasa/genética , Esfingomielina Fosfodiesterasa/metabolismo , Animales , Doxiciclina/farmacología , Eliminación de Gen , Expresión Génica , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Animales , Osteogénesis/efectos de los fármacos , Osteogénesis Imperfecta/genética
14.
Transl Neurodegener ; 12(1): 51, 2023 11 10.
Artículo en Inglés | MEDLINE | ID: mdl-37950283

RESUMEN

BACKGROUND: Intraneuronal accumulation of hyperphosphorylated tau is a defining hallmark of Alzheimer's disease (AD). However, mouse models imitating AD-exclusive neuronal tau pathologies are lacking. METHODS: We generated a new tet-on transgenic mouse model expressing truncated human tau N1-368 (termed hTau368), a tau fragment increased in the brains of AD patients and aged mouse brains. Doxycycline (dox) was administered in drinking water to induce hTau368 expression. Immunostaining and Western blotting were performed to measure the tau level. RNA sequencing was performed to evaluate gene expression, and several behavioral tests were conducted to evaluate mouse cognitive functions, emotion and locomotion. RESULTS: Dox treatment for 1-2 months at a young age induced overt and reversible human tau accumulation in the brains of hTau368 transgenic mice, predominantly in the hippocampus. Meanwhile, the transgenic mice exhibited AD-like high level of tau phosphorylation, glial activation, loss of mature neurons, impaired hippocampal neurogenesis, synaptic degeneration and cognitive deficits. CONCLUSIONS: This study developed a well-characterized and easy-to-use tool for the investigations and drug development for AD and other tauopathies.


Asunto(s)
Enfermedad de Alzheimer , Tauopatías , Animales , Humanos , Ratones , Enfermedad de Alzheimer/metabolismo , Hipocampo/metabolismo , Ratones Transgénicos , Proteínas tau/genética , Proteínas tau/metabolismo , Tauopatías/genética , Tauopatías/metabolismo , Tauopatías/patología
15.
ACS Synth Biol ; 12(2): 482-491, 2023 02 17.
Artículo en Inglés | MEDLINE | ID: mdl-36755406

RESUMEN

Synthetic expression cassettes provide the ability to control transgene expression in experimental animal models through external triggers, enabling the study of gene function and the modulation of endogenous regulatory networks in vivo. The performance of synthetic expression cassettes in transgenic animals critically depends on the regulatory properties of the respective chromosomal integration sites, which are affected by the remodeling of the chromatin structure during development. The epigenetic status may affect the transcriptional activity of the synthetic cassettes and even lead to transcriptional silencing, depending on the chromosomal sites and the tissue. In this study, we investigated the influence of the ubiquitous chromosome opening element (UCOE) HNRPA2B1-CBX3 and its subfragments A2UCOE and CBX3 on doxycycline-controlled expression modules within the chromosomal Rosa26 locus. While HNRPA2B1-CBX3 and A2UCOE reduced the expression of the synthetic cassettes in mouse embryonic stem cells, CBX3 stabilized the expression and facilitated doxycycline-controlled expression after in vitro differentiation. In transgenic mice, the CBX3 element protected the cassettes from overt silencing although the expression was moderate and only partially controlled by doxycycline. We demonstrate that CBX3-flanked synthetic cassettes can be activated by decitabine-mediated blockade of DNA methylation or by specific recruitment of the catalytic demethylation domain of the ten-eleven translocation protein TET1 to the synthetic promoter. This suggests that CBX3 renders the synthetic cassettes permissive for subsequent epigenetic activation, thereby supporting doxycycline-controlled expression. Together, this study reveals a strategy for overcoming epigenetic constraints of synthetic expression cassettes, facilitating externally controlled transgene expression in mice.


Asunto(s)
Cromatina , Doxiciclina , Ratones , Animales , Ratones Transgénicos , Doxiciclina/farmacología , Desmetilación del ADN , Transgenes/genética , Diferenciación Celular/genética
16.
Zhonghua Xue Ye Xue Za Zhi ; 44(5): 366-372, 2023 May 14.
Artículo en Zh | MEDLINE | ID: mdl-37550185

RESUMEN

Objective: To investigate the effect of the AML1-ETO (AE) fusion gene on the biological function of U937 leukemia cells by establishing a leukemia cell model that induces AE fusion gene expression. Methods: The doxycycline (Dox) -dependent expression of the AE fusion gene in the U937 cell line (U937-AE) were established using a lentivirus vector system. The Cell Counting Kit 8 methods, including the PI and sidanilide induction, were used to detect cell proliferation, cell cycle-induced differentiation assays, respectively. The effect of the AE fusion gene on the biological function of U937-AE cells was preliminarily explored using transcriptome sequencing and metabonomic sequencing. Results: ①The Dox-dependent Tet-on regulatory system was successfully constructed to regulate the stable AE fusion gene expression in U937-AE cells. ②Cell proliferation slowed down and the cell proliferation rate with AE expression (3.47±0.07) was lower than AE non-expression (3.86 ± 0.05) after inducing the AE fusion gene expression for 24 h (P<0.05). The proportion of cells in the G(0)/G(1) phase in the cell cycle increased, with AE expression [ (63.45±3.10) %) ] was higher than AE non-expression [ (41.36± 9.56) %] (P<0.05). The proportion of cells expressing CD13 and CD14 decreased with the expression of AE. The AE negative group is significantly higher than the AE positive group (P<0.05). ③The enrichment analysis of the transcriptome sequencing gene set revealed significantly enriched quiescence, nuclear factor kappa-light-chain-enhancer of activated B cells, interferon-α/γ, and other inflammatory response and immune regulation signals after AE expression. ④Disorder of fatty acid metabolism of U937-AE cells occurred under the influence of AE. The concentration of the medium and short-chain fatty acid acylcarnitine metabolites decreased in cells with AE expressing, propionyl L-carnitine, wherein those with AE expression (0.46±0.13) were lower than those with AE non-expression (1.00±0.27) (P<0.05). The metabolite concentration of some long-chain fatty acid acylcarnitine increased in cells with AE expressing tetradecanoyl carnitine, wherein those with AE expression (1.26±0.01) were higher than those with AE non-expression (1.00±0.05) (P<0.05) . Conclusion: This study successfully established a leukemia cell model that can induce AE expression. The AE expression blocked the cell cycle and inhibited cell differentiation. The gene sets related to the inflammatory reactions was significantly enriched in U937-AE cells that express AE, and fatty acid metabolism was disordered.


Asunto(s)
Leucemia Mieloide Aguda , Leucemia , Humanos , Células U937 , Proteína 1 Compañera de Translocación de RUNX1 , Leucemia/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Proteínas de Fusión Oncogénica/genética , Leucemia Mieloide Aguda/genética
17.
Sci China Life Sci ; 65(11): 2269-2286, 2022 11.
Artículo en Inglés | MEDLINE | ID: mdl-35596888

RESUMEN

Inducible expression systems are indispensable for precise regulation and in-depth analysis of biological process. Binary Tet-On system has been widely employed to regulate transgenic expression by doxycycline. Previous pig models with tetracycline regulatory elements were generated through random integration. This process often resulted in uncertain expression and unpredictable phenotypes, thus hindering their applications. Here, by precise knock-in of binary Tet-On 3G elements into Rosa26 and Hipp11 locus, respectively, a double knock-in reporter pig model was generated. We characterized excellent properties of this system for controllable transgenic expression both in vitro and in vivo. Two attP sites were arranged to flank the tdTomato to switch reporter gene. Single or multiple gene replacement was efficiently and faithfully achieved in fetal fibroblasts and nuclear transfer embryos. To display the flexible application of this system, we generated a pig strain with Dox-inducing hKRASG12D expression through phiC31 integrase-mediated cassette exchange. After eight months of Dox administration, squamous cell carcinoma developed in the nose, mouth, and scrotum, which indicated this pig strain could serve as an ideal large animal model to study tumorigenesis. Overall, the established pig models with controllable and switchable transgene expression system will provide a facilitating platform for transgenic and biomedical research.


Asunto(s)
Terapia Genética , Integrasas , Masculino , Animales , Porcinos , Integrasas/genética , Integrasas/metabolismo , Transgenes , Animales Modificados Genéticamente , Expresión Génica
18.
Cancer Lett ; 509: 26-38, 2021 07 01.
Artículo en Inglés | MEDLINE | ID: mdl-33819529

RESUMEN

Oncolytic adenovirus-mediated gene therapy shows promise for cancer treatment; however, the systemic delivery of oncolytic adenovirus to tumors remains challenging. Recently, mesenchymal stem cells (MSCs) have emerged as potential vehicles for improving delivery. Yet, because the oncolytic adenovirus replicates in MSCs, balancing MSC viability with viral load is key to achieving optimal therapeutic effect. We thus developed an all-in-one Tet-on system that can regulate replication of oncolytic adenovirus. Then, we loaded the novel oncolytic adenovirus carrying interleukin (IL)-24 and/or Endostatin in human umbilical cord blood-mesenchymal stem cells (hUCB-MSCs) for glioma therapy. In vitro assays demonstrated that this novel oncolytic adenovirus could efficiently replicate and kill glioma cells while sparing normal cells. Moreover, doxycycline effectively regulated oncolytic adenovirus replication in the hUCB-MSCs. The doxycycline induction group with dual expression of IL-24 and Endostatin exhibited significantly greater antitumor effects than other groups in a xenograft model of glioma. Thus, this strategy for systemic delivery of oncolytic adenovirus with its oncolytic activity controlled by a Tet-on system is a promising method for achieving antitumor efficacy in glioma, especially for metastatic tumors.


Asunto(s)
Neoplasias Encefálicas/terapia , Trasplante de Células Madre de Sangre del Cordón Umbilical , Endostatinas/biosíntesis , Terapia Genética , Glioma/terapia , Interleucinas/biosíntesis , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/virología , Viroterapia Oncolítica , Virus Oncolíticos/genética , Animales , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/virología , Muerte Celular , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Endostatinas/genética , Femenino , Vectores Genéticos , Glioma/genética , Glioma/metabolismo , Glioma/virología , Humanos , Interleucinas/genética , Ratones Endogámicos BALB C , Ratones Desnudos , Virus Oncolíticos/crecimiento & desarrollo , Carga Tumoral , Replicación Viral , Ensayos Antitumor por Modelo de Xenoinjerto
19.
Methods Mol Biol ; 2366: 193-212, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34236640

RESUMEN

Therapy-induced senescence (TIS or therapy-induced premature senescence) is a key cellular program triggered in the course of cancer radiotherapy and chemotherapy with genotoxic drugs, both in cancer cells and in normal cells, whose activation critically affects the outcome of cancer therapy. Drug-induced senescent cells undergo a permanent cell cycle arrest, acquire distinctive morphological and biochemical alterations, and an enhanced secretory ability, referred to as senescence-associated secretory phenotype (SASP). The transcription factor NF-κB acts as a master regulator of the SASP, driving the expression of senescence-associated secretome components.Here we describe protocols for the establishment of a tetracycline-regulated cell system for the investigation of the role of NF-κB in TIS. We also describe protocols routinely used in our laboratory, to investigate TIS in this Tet-On inducible expression system. Finally, we describe techniques for the validation of TIS induction.


Asunto(s)
Senescencia Celular , Antineoplásicos/farmacología , Senescencia Celular/efectos de los fármacos , FN-kappa B/genética , FN-kappa B/metabolismo , Inhibidores de la Síntesis de la Proteína/farmacología , Secretoma , Fenotipo Secretor Asociado a la Senescencia , Tetraciclina/farmacología
20.
Insects ; 11(11)2020 Nov 11.
Artículo en Inglés | MEDLINE | ID: mdl-33187095

RESUMEN

The Pv11 cell line established from an African chironomid, Polypedilum vanderplanki, is the only cell line tolerant to complete desiccation. In Pv11 cells, a constitutive expression system for Pv11 cells was previously exploited and several reporter genes were successfully expressed. Here we report the identification of an effective minimal promoter for Pv11 cells and its application to the Tet-On inducible expression system. First, using a luciferase reporter assay, we showed that a 202 bp deletion fragment derived from the constitutively active 121-promoter functions in Pv11 cells as an appropriate minimal promoter with the Tet-On inducible expression system. The AcGFP1 (Aequorea coerulescens green fluorescent protein) was also successfully expressed in Pv11 cells using the inducible system. In addition to these reporter genes, the avian myeloblastosis virus reverse transcriptase α subunit (AMV RTα), which is one of the most widely commercially available RNA-dependent DNA polymerases, was successfully expressed through the inducible expression system and its catalytic activity was verified. These results demonstrate the establishment of an inducible expression system in cells that can be preserved in the dry state and highlight a possible application to the production of large and complex proteins.

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