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1.
Int J Mol Sci ; 22(6)2021 Mar 16.
Artículo en Inglés | MEDLINE | ID: mdl-33809617

RESUMEN

This study aimed to identify alternative anti-inflammatory compounds that modulate the activity of a relevant transcription factor, CCAAT/enhancer binding protein delta (C/EBPδ). C/EBPδ is a master regulator of inflammatory responses in macrophages (Mϕ) and is mainly regulated at the level of CEBPD gene transcription initiation. To screen for CEBPD-modulating compounds, we generated a THP-1-derived reporter cell line stably expressing secreted alkaline phosphatase (SEAP) under control of the defined CEBPD promoter (CEBPD::SEAP). A high-throughput screening of LOPAC®1280 and ENZO®774 libraries on LPS- and IFN-γ-activated THP-1 reporter Mϕ identified four epigenetically active hits: two bromodomain and extraterminal domain (BET) inhibitors, I-BET151 and Ro 11-1464, as well as two histone deacetylase (HDAC) inhibitors, SAHA and TSA. All four hits markedly and reproducibly upregulated SEAP secretion and CEBPD::SEAP mRNA expression, confirming screening assay reliability. Whereas BET inhibitors also upregulated the mRNA expression of the endogenous CEBPD, HDAC inhibitors completely abolished it. All hits displayed anti-inflammatory activity through the suppression of IL-6 and CCL2 gene expression. However, I-BET151 and HDAC inhibitors simultaneously upregulated the mRNA expression of pro-inflammatory IL-1ß. The modulation of CEBPD gene expression shown in this study contributes to our understanding of inflammatory responses in Mϕ and may offer an approach to therapy for inflammation-driven disorders.


Asunto(s)
Antiinflamatorios/farmacología , Proteína delta de Unión al Potenciador CCAAT/metabolismo , Genes Reporteros , Ensayos Analíticos de Alto Rendimiento , Inhibidores de Histona Desacetilasas/farmacología , Macrófagos/metabolismo , Fosfatasa Alcalina/metabolismo , Azepinas/farmacología , Proteína delta de Unión al Potenciador CCAAT/antagonistas & inhibidores , Regulación de la Expresión Génica/efectos de los fármacos , Células HEK293 , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Humanos , Ácidos Hidroxámicos/farmacología , Mediciones Luminiscentes , Macrófagos/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Células THP-1 , Tiofenos/farmacología , Vorinostat/farmacología
2.
Int J Mol Sci ; 22(5)2021 Mar 05.
Artículo en Inglés | MEDLINE | ID: mdl-33807548

RESUMEN

About 8% of the human genome is covered with candidate cis-regulatory elements (cCREs). Disruptions of CREs, described as "cis-ruptions" have been identified as being involved in various genetic diseases. Thanks to the development of chromatin conformation study techniques, several long-range cystic fibrosis transmembrane conductance regulator (CFTR) regulatory elements were identified, but the regulatory mechanisms of the CFTR gene have yet to be fully elucidated. The aim of this work is to improve our knowledge of the CFTR gene regulation, and to identity factors that could impact the CFTR gene expression, and potentially account for the variability of the clinical presentation of cystic fibrosis as well as CFTR-related disorders. Here, we apply the robust GWAS3D score to determine which of the CFTR introns could be involved in gene regulation. This approach highlights four particular CFTR introns of interest. Using reporter gene constructs in intestinal cells, we show that two new introns display strong cooperative effects in intestinal cells. Chromatin immunoprecipitation analyses further demonstrate fixation of transcription factors network. These results provide new insights into our understanding of the CFTR gene regulation and allow us to suggest a 3D CFTR locus structure in intestinal cells. A better understand of regulation mechanisms of the CFTR gene could elucidate cases of patients where the phenotype is not yet explained by the genotype. This would thus help in better diagnosis and therefore better management. These cis-acting regions may be a therapeutic challenge that could lead to the development of specific molecules capable of modulating gene expression in the future.


Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Intestinos/fisiología , Células CACO-2 , Línea Celular Tumoral , Inmunoprecipitación de Cromatina/métodos , Fibrosis Quística/genética , Regulación de la Expresión Génica/genética , Genes Reporteros/genética , Humanos , Intrones/genética , Factores de Transcripción/genética
3.
Int J Mol Sci ; 22(6)2021 Mar 11.
Artículo en Inglés | MEDLINE | ID: mdl-33799598

RESUMEN

We sought to develop a cell-based cytotoxicity assay using human hepatocytes, which reflect the effects of drug-metabolizing enzymes on cytotoxicity. In this study, we generated luminescent human hepatoblastoma HepG2 cells using the mouse artificial chromosome vector, in which click beetle luciferase alone or luciferase and major drug-metabolizing enzymes (CYP2C9, CYP2C19, CYP2D6, and CYP3A4) are expressed, and monitored the time-dependent changes of CYP-mediated cytotoxicity expression by bioluminescence measurement. Real-time bioluminescence measurement revealed that compared with CYP-non-expressing cells, the luminescence intensity of CYP-expressing cells rapidly decreased when the cells were treated with low concentrations of aflatoxin B1 or primaquine, which exhibits cytotoxicity in the presence of CYP3A4 or CYP2D6, respectively. Using kinetics data obtained by the real-time bioluminescence measurement, we estimated the time-dependent changes of 50% inhibitory concentration (IC50) values in the aflatoxin B1- and primaquine-treated cell lines. The first IC50 value was detected much earlier and at a lower concentration in primaquine-treated CYP-expressing HepG2 cells than in primaquine-treated CYP-non-expressing cells, and the decrease of IC50 values was much faster in the former than the latter. Thus, we successfully monitored time- and concentration-dependent dynamic changes of CYP-mediated cytotoxicity expression in CYP-expressing luminescent HepG2 cells by means of real-time bioluminescence measurement.


Asunto(s)
Aflatoxina B1/toxicidad , Efecto Fundador , Mediciones Luminiscentes/métodos , Primaquina/toxicidad , Imagen de Lapso de Tiempo/métodos , Xenobióticos/toxicidad , Animales , Línea Celular Tumoral , Citocromo P-450 CYP2C19/genética , Citocromo P-450 CYP2C19/metabolismo , Citocromo P-450 CYP2C9/genética , Citocromo P-450 CYP2C9/metabolismo , Citocromo P-450 CYP2D6/genética , Citocromo P-450 CYP2D6/metabolismo , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Regulación de la Expresión Génica , Genes Reporteros , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Células Hep G2 , Humanos , Concentración 50 Inhibidora , Luciferasas/genética , Luciferasas/metabolismo , Luminiscencia , Ratones
4.
Cancer Invest ; 39(4): 285-296, 2021 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-33646061

RESUMEN

The switchable chimeric antigen receptors (CARs) have shown many advantages in CAR T-cell therapy. However, human primary T-cells are required to evaluate antigen-specific adaptors by IFN-γ assay or FACS analysis, which limits the throughput of adaptor screening. A sensitive and robust CD16-CAR Jurkat NFAT-eGFP reporter system has been developed to assess the therapeutic efficacy of antibody-targeted CAR-T-cell by effectively evaluating the T-cell activation by various tumor cells and the impact of immune checkpoint inhibitor antibodies. This reporter system facilitates the screening of targeted antibodies in a high throughput manner for the development of improved T-cell immunotherapy.


Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Antineoplásicos Inmunológicos/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Antígeno B7-H1/antagonistas & inhibidores , Cetuximab/farmacología , Inmunoterapia Adoptiva , Neoplasias/terapia , Receptores Quiméricos de Antígenos/inmunología , Receptores de IgG/inmunología , Linfocitos T/trasplante , Células A549 , Antígeno B7-H1/inmunología , Antígeno B7-H1/metabolismo , Ensayos de Selección de Medicamentos Antitumorales , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/inmunología , Receptores ErbB/metabolismo , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/inmunología , Proteínas Ligadas a GPI/metabolismo , Genes Reporteros , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Células HCT116 , Ensayos Analíticos de Alto Rendimiento , Humanos , Células Jurkat , Factores de Transcripción NFATC/genética , Neoplasias/genética , Neoplasias/inmunología , Neoplasias/metabolismo , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/metabolismo , Receptores de IgG/genética , Receptores de IgG/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo
5.
Int J Mol Sci ; 22(4)2021 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-33671852

RESUMEN

SINE-VNTR-Alu (SVA) retrotransposons are a subclass of transposable elements (TEs) that exist only in primate genomes. TE insertions can be co-opted as cis-regulatory elements (CREs); however, the regulatory potential of SVAs has predominantly been demonstrated using bioinformatic approaches and reporter gene assays. The objective of this study was to demonstrate SVA cis-regulatory activity by CRISPR (clustered regularly interspaced short palindromic repeats) deletion and subsequent measurement of direct effects on local gene expression. We identified a region on chromosome 17 that was enriched with human-specific SVAs. Comparative gene expression analysis at this region revealed co-expression of TRPV1 and TRPV3 in multiple human tissues, which was not observed in mouse, highlighting key regulatory differences between the two species. Furthermore, the intergenic region between TRPV1 and TRPV3 coding sequences contained a human specific SVA insertion located upstream of the TRPV3 promoter and downstream of the 3' end of TRPV1, highlighting this SVA as a candidate to study its potential cis-regulatory activity on both genes. Firstly, we generated SVA reporter gene constructs and demonstrated their transcriptional regulatory activity in HEK293 cells. We then devised a dual-targeting CRISPR strategy to facilitate the deletion of this entire SVA sequence and generated edited HEK293 clonal cell lines containing homozygous and heterozygous SVA deletions. In edited homozygous ∆SVA clones, we observed a significant decrease in both TRPV1 and TRPV3 mRNA expression, compared to unedited HEK293. In addition, we also observed an increase in the variability of mRNA expression levels in heterozygous ∆SVA clones. Overall, in edited HEK293 with SVA deletions, we observed a disruption to the co-expression of TRPV1 and TRPV3. Here we provide an example of a human specific SVA with cis-regulatory activity in situ, supporting the role of SVA retrotransposons as contributors to species-specific gene expression.


Asunto(s)
Elementos Alu/genética , Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , ADN Intergénico/genética , Repeticiones de Minisatélite/genética , Regiones Promotoras Genéticas/genética , Elementos de Nucleótido Esparcido Corto/genética , Canales Catiónicos TRPV/genética , Animales , Expresión Génica , Regulación de la Expresión Génica , Genes Reporteros , Células HEK293 , Humanos , Ratones , Primates/genética
6.
Nat Commun ; 12(1): 1955, 2021 03 29.
Artículo en Inglés | MEDLINE | ID: mdl-33782410

RESUMEN

p62/SQSTM1 is known to act as a key mediator in the selective autophagy of protein aggregates, or aggrephagy, by steering ubiquitinated protein aggregates towards the autophagy pathway. Here, we use a yeast two-hybrid screen to identify the prefoldin-like chaperone UXT as an interacting protein of p62. We show that UXT can bind to protein aggregates as well as the LB domain of p62, and, possibly by forming an oligomer, increase p62 clustering for its efficient targeting to protein aggregates, thereby promoting the formation of the p62 body and clearance of its cargo via autophagy. We also find that ectopic expression of human UXT delays SOD1(A4V)-induced degeneration of motor neurons in a Xenopus model system, and that specific disruption of the interaction between UXT and p62 suppresses UXT-mediated protection. Together, these results indicate that UXT functions as an autophagy adaptor of p62-dependent aggrephagy. Furthermore, our study illustrates a cooperative relationship between molecular chaperones and the aggrephagy machinery that efficiently removes misfolded protein aggregates.


Asunto(s)
Autofagia/genética , Proteínas de Ciclo Celular/genética , Chaperonas Moleculares/genética , Agregado de Proteínas , Proteína Sequestosoma-1/genética , Superóxido Dismutasa-1/genética , Animales , Autofagia/efectos de los fármacos , Proteínas de Ciclo Celular/metabolismo , Ganglios Espinales/citología , Ganglios Espinales/metabolismo , Regulación de la Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Células HeLa , Humanos , Leupeptinas/farmacología , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Chaperonas Moleculares/metabolismo , Neuronas Motoras/citología , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/metabolismo , Cultivo Primario de Células , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/metabolismo , Agregado de Proteínas/efectos de los fármacos , Pliegue de Proteína/efectos de los fármacos , Proteína Sequestosoma-1/metabolismo , Transducción de Señal , Superóxido Dismutasa-1/metabolismo , Transgenes , Xenopus laevis
7.
J Vis Exp ; (168)2021 02 26.
Artículo en Inglés | MEDLINE | ID: mdl-33720134

RESUMEN

Age-related macular degeneration (AMD) is the most frequent cause of blindness in patients >60 years, affecting ~30 million people worldwide. AMD is a multifactorial disease influenced by environmental and genetic factors, which lead to functional impairment of the retina due to retinal pigment epithelial (RPE) cell degeneration followed by photoreceptor degradation. An ideal treatment would include the transplantation of healthy RPE cells secreting neuroprotective factors to prevent RPE cell death and photoreceptor degeneration. Due to the functional and genetic similarities and the possibility of a less invasive biopsy, the transplantation of iris pigment epithelial (IPE) cells was proposed as a substitute for the degenerated RPE. Secretion of neuroprotective factors by a low number of subretinally-transplanted cells can be achieved by Sleeping Beauty (SB100X) transposon-mediated transfection with genes coding for the pigment epithelium-derived factor (PEDF) and/or the granulocyte macrophage-colony stimulating factor (GM-CSF). We established the isolation, culture, and SB100X-mediated transfection of RPE and IPE cells from various species including rodents, pigs, and cattle. Globes are explanted and the cornea and lens are removed to access the iris and the retina. Using a custom-made spatula, IPE cells are removed from the isolated iris. To harvest RPE cells, a trypsin incubation may be required, depending on the species. Then, using RPE-customized spatula, cells are suspended in medium. After seeding, cells are monitored twice per week and, after reaching confluence, transfected by electroporation. Gene integration, expression, protein secretion, and function were confirmed by qPCR, WB, ELISA, immunofluorescence, and functional assays. Depending on the species, 30,000-5 million (RPE) and 10,000-1.5 million (IPE) cells can be isolated per eye. Genetically modified cells show significant PEDF/GM-CSF overexpression with the capacity to reduce oxidative stress and offers a flexible system for ex vivo analyses and in vivo studies transferable to humans to develop ocular gene therapy approaches.


Asunto(s)
Separación Celular/métodos , Ingeniería Genética , Terapia Genética , Mamíferos/metabolismo , Epitelio Pigmentado de la Retina/citología , Animales , Bovinos , Supervivencia Celular , Células Cultivadas , Electroporación , Proteínas del Ojo/genética , Proteínas del Ojo/uso terapéutico , Genes Reporteros , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Factor Estimulante de Colonias de Granulocitos y Macrófagos/uso terapéutico , Ratones , Factores de Crecimiento Nervioso/genética , Factores de Crecimiento Nervioso/uso terapéutico , Estrés Oxidativo/genética , Ratas , Serpinas/genética , Serpinas/uso terapéutico , Porcinos , Transfección
8.
Nat Commun ; 12(1): 1846, 2021 03 23.
Artículo en Inglés | MEDLINE | ID: mdl-33758180

RESUMEN

A wide repertoire of genetic switches has accelerated prokaryotic synthetic biology, while eukaryotic synthetic biology has lagged in the model organism Saccharomyces cerevisiae. Eukaryotic genetic switches are larger and more complex than prokaryotic ones, complicating the rational design and evolution of them. Here, we present a robust workflow for the creation and evolution of yeast genetic switches. The selector system was designed so that both ON- and OFF-state selection of genetic switches is completed solely by liquid handling, and it enabled parallel screen/selection of different motifs with different selection conditions. Because selection threshold of both ON- and OFF-state selection can be flexibly tuned, the desired selection conditions can be rapidly pinned down for individual directed evolution experiments without a prior knowledge either on the library population. The system's utility was demonstrated using 20 independent directed evolution experiments, yielding genetic switches with elevated inducer sensitivities, inverted switching behaviours, sensory functions, and improved signal-to-noise ratio (>100-fold induction). The resulting yeast genetic switches were readily integrated, in a plug-and-play manner, into an AND-gated carotenoid biosynthesis pathway.


Asunto(s)
Evolución Molecular Dirigida/métodos , Genes de Cambio , Ingeniería Genética/métodos , Técnicas Genéticas , Saccharomyces cerevisiae/genética , Biología Sintética/métodos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Basidiomycota/genética , Basidiomycota/metabolismo , Citometría de Flujo , Biblioteca de Genes , Genes Reporteros , Floroglucinol/análogos & derivados , Floroglucinol/farmacología , Regiones Promotoras Genéticas , Proteínas Represoras/química , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Relación Señal-Ruido , Tetraciclina/farmacología , Transactivadores/química , Transactivadores/genética , Transactivadores/metabolismo , beta Caroteno/biosíntesis , beta Caroteno/genética , beta Caroteno/metabolismo
9.
Sci Total Environ ; 771: 144514, 2021 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-33736142

RESUMEN

Every year thousands of chemicals get discharged into the waterbodies of the world. These chemicals cause endocrine disruption and induce adverse health effects in human and aquatic life. Global environmental protection agencies emphasise the need to develop rapid and specific tests for identification of these endocrine disruptive chemicals (EDCs) in water. Detection of chemicals that disrupt androgen signaling is especially important because androgen input at specific phases of life is critical for proper male development. Effect-based methods such as reporter assays are suitable tools for identification of EDCs in mixtures of unknown composition. The current study describes a stable, secreted alkaline protease (SEAP)-based reporter assay system, for visual detection of androgenic/antiandrogenic activity present in water samples. A novel feature of this system is the inclusion of coactivators, GRIP1, CARM1, p300 and mZac1b, in addition to an optimal combination of androgen response element (3× HRE), androgen receptor (AR) and the SEAP reporter gene. Incorporation of the coactivators resulted in a transcriptional fold change of 162 folds, enabling visual detection at much lower concentrations of androgen (1 picomolar) within 1 h of addition of test sample. Also, non-androgenic steroids such as estrogen, progesterone and Dexamethasone did not induce significant reporter activity, except at very high concentrations. This reporter assay can be readily converted into a high throughput format for investigation in multiple samples simultaneously, and reflects the changes that can be expected to occur inside a mammalian cell. The androgenic activity in six different water sources was evaluated using this assay. The results reveal significant androgenic activity in rivers and lakes close to Industrial areas, whereas the highest androgenic activity was observed in water containing paper and pulp mill effluents. This bioassay therefore provides a rapid, visual detection tool for effect-directed analysis of androgenic/antiandrogenic compounds in water. IMPACT STATEMENT: The current SEAP-based assay allows visual detection of androgens/antiandrogens in water, at concentrations as low as 1 picomolar, within a 1 h time period, in a high throughput format, providing a very useful technique for field users and regulatory bodies.


Asunto(s)
Antagonistas de Andrógenos , Andrógenos , Antagonistas de Andrógenos/toxicidad , Animales , Proteínas Bacterianas , Bioensayo , Endopeptidasas , Genes Reporteros , Humanos , Masculino , Agua
10.
Sheng Wu Gong Cheng Xue Bao ; 37(2): 655-662, 2021 Feb 25.
Artículo en Chino | MEDLINE | ID: mdl-33645163

RESUMEN

The transposon vector containing enhanced green fluorescent protein (EGFP) was injected into early housefly (Musca domestica L.) eggs by microinjection method to realize stable gene expression in vivo for verification, and to study housefly gene function. A borosilicate glass micro injection needle suitable for microinjection of housefly eggs was made, the softening treatment conditions of housefly egg shells were explored, and a microinjection technology platform suitable for housefly was constructed with a high-precision microsyringe Nanoject Ⅲ as the main body. The recombinant plasmid PiggyBac-[3×P3]-EGFP containing the eye-specific 3×P3 promoter and EGFP and the stable genetic expression helper plasmid pHA3pig helper were microinjected into the treated housefly eggs. After emergence, the eye luminescence was observed, and the expression and transcription level of EGFP were detected. The results showed that the normal hatching rate of housefly eggs was 55% when rinsed in bleaching water for 35 s. The hardness of the egg shell treated for 35 s was suitable for injection and the injection needle was not easy to break. About 3% of the emerged housefly eyes had green fluorescence. Through further molecular detection, EGFP specific fragments with a size of 750 bp were amplified from DNA and RNA of housefly. Through the technical platform, the stable expression of reporter genes in housefly can be conveniently and effectively realized, and a bioreactor with housefly as the main body can be established, which provides certain reference value for subsequent research on housefly gene function.


Asunto(s)
Moscas Domésticas , Animales , Animales Modificados Genéticamente , Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Moscas Domésticas/genética , Microinyecciones
11.
Methods Mol Biol ; 2224: 1-27, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33606203

RESUMEN

Recent development of Easi-CRISPR (Efficient additions with ssDNA inserts-CRISPR) that utilizes long single-stranded DNA (lssDNA) of 0.2-2 kbases in length as donor templates to insert large segments of novel DNA sequences or to replace endogenous genes at precise locations in the genome has enabled CRISPR-assisted genome editing to make strides toward a more simple and rapid workflow. By leveraging the notion that short single-stranded DNA oligo (<200 bases) serves as efficient donor in mouse zygotes for facilitating HDR-mediated genome editing, Easi-CRISPR expands to use lssDNA as the donor which accelerates the timeline to as little as 2 months for creating most types of genetically engineered mouse models (F0). Our lab (CGERC) has adopted Easi-CRISPR for multiple loci to generate mouse models over the past three plus years since its introduction. Here, we use two genes as examples to illustrate a step-by-step protocol for generating two commonly used models, including a knock-in (insertion of a reporter gene plus GOI) as well as a conditional knock-out model (via exon floxing). This protocol will focus more on molecular biology aspect, particularly we demonstrate two recently developed methods for lssDNA procuration: (1) PCR-based Takara Bio kit with modifications; (2) plasmid-retrieval-based CRISPR-CLIP (CRISPR-Clipped LssDNA via Incising Plasmid). Both methods are devised to retain sequence fidelity in lssDNA generated. In addition, CRISPR-CLIP directly retrieves lssDNA from DNA plasmid without using restriction enzymes through a PCR-free system hence carries virtually no restriction on sequence complexity, further mitigating limitations discussed in the original Easi-CRISPR protocol. We have alternated the use between both methods when suitable and successfully generated lssDNA templates via CRISPR-CLIP up to 3.5 kbases patched with multiple highly repetitive sequences, which is otherwise challenging to maneuver. Along with certain other modified workflow presented herein, Easi-CRISPR can be adapted to be more straightforward while applicable to generate mouse models in broader scope. (Certain figures and text passages presented in this chapter are reproduced from Shola et al. (The CRISPR J 3(2):109-122, 2020), published by Mary Ann Libert, Inc).


Asunto(s)
Sistemas CRISPR-Cas/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , ADN de Cadena Simple/genética , Animales , Exones/genética , Femenino , Edición Génica/métodos , Técnicas de Sustitución del Gen , Genes Reporteros/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Animales , ARN Guia/genética , Cigoto/fisiología
12.
Methods Mol Biol ; 2224: 29-45, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33606204

RESUMEN

Reporter mice transgenically expressing the bacterial (E. coli) lacZ gene encoding ß-galactosidase (ß-gal, EC 3.2.1.23) are a versatile and extensively used tool to study gene expression and cell lineage patterns. Enzymatic activity of the ß-gal reporter can be effectively visualized at cellular resolution either histochemically using 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-gal) or by immunofluorescent detection using a ß-gal-specific antibody. Here, we summarize protocols for the localization of ß-gal expressing cells in whole embryos or organs as well as in histological tissue sections of lacZ reporter mice and discuss their limitations and common pitfalls.


Asunto(s)
Expresión Génica/genética , Genes Reporteros/genética , Operón Lac/genética , Animales , Linaje de la Célula/genética , Embrión de Mamíferos/metabolismo , Escherichia coli/genética , Galactósidos/genética , Indoles , Ratones , Coloración y Etiquetado/métodos
13.
Methods Mol Biol ; 2224: 153-182, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33606214

RESUMEN

Hematopoiesis in the mouse and other mammals occurs in several waves and arises from distinct anatomic sites. Transgenic mice expressing fluorescent reporter proteins at various points in the hematopoietic hierarchy, from hematopoietic stem cell to more restricted progenitors to each of the final differentiated cell types, have provided valuable tools for tagging, tracking, and isolating these cells. In this chapter, we discuss general considerations in designing a transgene, survey available fluorescent probes, and describe methods for confirming and analyzing transgene expression in the hematopoietic tissues of the embryo, fetus, and postnatal/adult animal.


Asunto(s)
Genes Reporteros/genética , Hematopoyesis/genética , Proteínas Luminiscentes/genética , Animales , Diferenciación Celular/genética , Embrión de Mamíferos/fisiología , Femenino , Feto/fisiología , Células Madre Hematopoyéticas/fisiología , Masculino , Ratones , Ratones Transgénicos , Células Madre/fisiología , Transgenes/genética
14.
Cell Prolif ; 54(4): e13014, 2021 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-33615615

RESUMEN

INTRODUCTION: In vivo, cells are surrounded by extracellular matrix (ECM). To build organs from single cells, it is generally believed that ECM serves as scaffolds to coordinate cell positioning and differentiation. Nevertheless, how cells utilize cell-ECM interactions for the spatiotemporal coordination to different ECM at the tissue scale is not fully understood. METHODS: Here, using in vitro assay with engineered MDCK cells expressing H2B-mCherry (nucleus) and gp135/Podocalyxin-GFP (apical marker), we show in multi-dimensions that such coordination for epithelial morphogenesis can be determined by cell-soluble ECM interaction in the fluidic phase. RESULTS: The coordination depends on the native topology of ECM components such as sheet-like basement membrane (BM) and type I collagen (COL) fibres: scaffold formed by BM (COL) facilitates a close-ended (open-ended) coordination that leads to the formation of lobular (tubular) epithelium. Further, cells form apicobasal polarity throughout the entire lobule/tubule without a complete coverage of ECM at the basal side, and time-lapse two-photon scanning imaging reveals the polarization occurring early and maintained through the lobular expansion. During polarization, gp135-GFP was converged to the apical surface collectively in the lobular/tubular structures, suggesting possible intercellular communications. Under suspension culture, the polarization was impaired with multi-lumen formation in the tubules, implying the importance of ECM biomechanical microenvironment. CONCLUSION: Our results suggest a biophysical mechanism for cells to form polarity and coordinate positioning at tissue scale, and in engineering epithelium through cell-soluble ECM interaction and self-assembly.


Asunto(s)
Membrana Celular/metabolismo , Matriz Extracelular/metabolismo , Animales , Núcleo Celular/metabolismo , Polaridad Celular/fisiología , Colágeno Tipo I/metabolismo , Perros , Geles/química , Genes Reporteros , Células de Riñón Canino Madin Darby/citología , Células de Riñón Canino Madin Darby/metabolismo , Microscopía Fluorescente
15.
Nat Commun ; 12(1): 874, 2021 02 08.
Artículo en Inglés | MEDLINE | ID: mdl-33558533

RESUMEN

Base-pairing interactions mediate many intermolecular target recognition events. Even a single base-pair mismatch can cause a substantial difference in activity but how such changes influence the target search kinetics in vivo is unknown. Here, we use high-throughput sequencing and quantitative super-resolution imaging to probe the mutants of bacterial small RNA, SgrS, and their regulation of ptsG mRNA target. Mutations that disrupt binding of a chaperone protein, Hfq, and are distal to the mRNA annealing region still decrease the rate of target association, kon, and increase the dissociation rate, koff, showing that Hfq directly facilitates sRNA-mRNA annealing in vivo. Single base-pair mismatches in the annealing region reduce kon by 24-31% and increase koff by 14-25%, extending the time it takes to find and destroy the target by about a third. The effects of disrupting contiguous base-pairing are much more modest than that expected from thermodynamics, suggesting that Hfq buffers base-pair disruptions.


Asunto(s)
Emparejamiento Base/genética , Estabilidad del ARN , ARN Bacteriano/genética , Secuencia de Bases , Escherichia coli/genética , Dosificación de Gen , Genes Reporteros , Imagenología Tridimensional , Cinética , Mutación/genética , Nucleótidos/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Tiempo
16.
Mol Cell ; 81(5): 1013-1026.e11, 2021 03 04.
Artículo en Inglés | MEDLINE | ID: mdl-33548202

RESUMEN

In response to stress, human cells coordinately downregulate transcription and translation of housekeeping genes. To downregulate transcription, the negative elongation factor (NELF) is recruited to gene promoters impairing RNA polymerase II elongation. Here we report that NELF rapidly forms nuclear condensates upon stress in human cells. Condensate formation requires NELF dephosphorylation and SUMOylation induced by stress. The intrinsically disordered region (IDR) in NELFA is necessary for nuclear NELF condensation and can be functionally replaced by the IDR of FUS or EWSR1 protein. We find that biomolecular condensation facilitates enhanced recruitment of NELF to promoters upon stress to drive transcriptional downregulation. Importantly, NELF condensation is required for cellular viability under stressful conditions. We propose that stress-induced NELF condensates reported here are nuclear counterparts of cytosolic stress granules. These two stress-inducible condensates may drive the coordinated downregulation of transcription and translation, likely forming a critical node of the stress survival strategy.


Asunto(s)
Respuesta al Choque Térmico/genética , Proteínas Intrínsecamente Desordenadas/genética , Procesamiento Proteico-Postraduccional , ARN Polimerasa II/genética , Transcripción Genética , Factores de Elongación Transcripcional/genética , Aminoaciltransferasas/genética , Aminoaciltransferasas/metabolismo , Cromatina/química , Cromatina/metabolismo , Células Clonales , Quinasa 9 Dependiente de la Ciclina/genética , Quinasa 9 Dependiente de la Ciclina/metabolismo , Genes Reporteros , Células HEK293 , Células HeLa , Humanos , Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Fosforilación , Factor B de Elongación Transcripcional Positiva/genética , Factor B de Elongación Transcripcional Positiva/metabolismo , Regiones Promotoras Genéticas , ARN Polimerasa II/metabolismo , Transducción de Señal , Estrés Fisiológico , Sumoilación , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factores de Elongación Transcripcional/química , Factores de Elongación Transcripcional/metabolismo
17.
Toxicol Lett ; 342: 20-25, 2021 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-33581288

RESUMEN

Many bony features of the face develop from endochondral ossification of preexisting collagen-rich cartilage structures. The proper development of these cartilage structures is essential to the morphological formation of the face. The developmental programs governing the formation of the pre-bone facial cartilages are sensitive to chemical compounds that disturb histone acetylation patterns and chromatin structure. We have taken advantage of this fact to develop a quantitative morphological assay of craniofacial developmental toxicity based on the distortion and deterioration of facial cartilage structures in zebrafish larvae upon exposure to increasing concentrations of several well-described histone deacetylase inhibitors. In this assay, we measure the angle formed by the developing ceratohyal bone as a precise, sensitive and quantitative proxy for the overall developmental status of facial cartilages. Using the well-established developmental toxicant and histone deacetylase-inhibiting compound valproic acid along with 12 structurally related compounds, we demonstrate the applicability of the ceratohyal angle assay to investigate structure-activity relationships.


Asunto(s)
Butiratos/toxicidad , Colágeno Tipo II/metabolismo , Anomalías Craneofaciales/inducido químicamente , Histona Desacetilasas/metabolismo , Ácidos Hidroxámicos/toxicidad , Péptidos/toxicidad , Animales , Animales Modificados Genéticamente , Antibióticos Antineoplásicos/toxicidad , Anticonvulsivantes/toxicidad , Antifúngicos/toxicidad , Colágeno Tipo II/genética , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Genes Reporteros , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ácido Valproico/toxicidad , Pez Cebra
18.
Sensors (Basel) ; 21(3)2021 Jan 29.
Artículo en Inglés | MEDLINE | ID: mdl-33572727

RESUMEN

In recent years, there has been an increasing demand for predictive and sensitive in vitro tools for drug discovery. Split complementation assays have the potential to enlarge the arsenal of in vitro tools for compound screening, with most of them relying on well-established reporter gene assays. In particular, ligand-induced complementation of split luciferases is emerging as a suitable approach for monitoring protein-protein interactions. We hereby report an intracellular nanosensor for the screening of compounds with androgenic activity based on a split NanoLuc reporter. We also confirm the suitability of using 3D spheroids of Human Embryonic Kidney (HEK-293) cells for upgrading the 2D cell-based assay. A limit of detection of 4 pM and a half maximal effective concentration (EC50) of 1.7 ± 0.3 nM were obtained for testosterone with HEK293 spheroids. This genetically encoded nanosensor also represents a new tool for real time imaging of the activation state of the androgen receptor, thus being suitable for analysing molecules with androgenic activity, including new drugs or endocrine disrupting molecules.


Asunto(s)
Andrógenos , Mediciones Luminiscentes , Nanotecnología , Receptores Androgénicos , Genes Reporteros , Células HEK293 , Humanos , Luciferasas/genética , Receptores Androgénicos/genética
19.
Nat Commun ; 12(1): 841, 2021 02 05.
Artículo en Inglés | MEDLINE | ID: mdl-33547291

RESUMEN

A new life begins with the unification of the maternal and paternal chromosomes upon fertilization. The parental chromosomes first become enclosed in two separate pronuclei near the surface of the fertilized egg. The mechanisms that then move the pronuclei inwards for their unification are only poorly understood in mammals. Here, we report two mechanisms that act in concert to unite the parental genomes in fertilized mouse eggs. The male pronucleus assembles within the fertilization cone and is rapidly moved inwards by the flattening cone. Rab11a recruits the actin nucleation factors Spire and Formin-2 into the fertilization cone, where they locally nucleate actin and further accelerate the pronucleus inwards. In parallel, a dynamic network of microtubules assembles that slowly moves the male and female pronuclei towards the cell centre in a dynein-dependent manner. Both mechanisms are partially redundant and act in concert to unite the parental pronuclei in the zygote's centre.


Asunto(s)
Núcleo Celular/metabolismo , Fertilización/genética , Forminas/genética , Proteínas de Microfilamentos/genética , Proteínas del Tejido Nervioso/genética , Cigoto/metabolismo , Proteínas de Unión al GTP rab/genética , Actinas/genética , Actinas/metabolismo , Animales , Núcleo Celular/ultraestructura , Femenino , Forminas/metabolismo , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Proteínas de Microfilamentos/metabolismo , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Movimiento , Proteínas del Tejido Nervioso/metabolismo , Oocitos/metabolismo , Oocitos/ultraestructura , Espermatozoides/metabolismo , Espermatozoides/ultraestructura , Cigoto/ultraestructura , Proteínas de Unión al GTP rab/metabolismo
20.
RNA ; 27(4): 445-464, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33397688

RESUMEN

Pumilio paralogs, PUM1 and PUM2, are sequence-specific RNA-binding proteins that are essential for vertebrate development and neurological functions. PUM1&2 negatively regulate gene expression by accelerating degradation of specific mRNAs. Here, we determined the repression mechanism and impact of human PUM1&2 on the transcriptome. We identified subunits of the CCR4-NOT (CNOT) deadenylase complex required for stable interaction with PUM1&2 and to elicit CNOT-dependent repression. Isoform-level RNA sequencing revealed broad coregulation of target mRNAs through the PUM-CNOT repression mechanism. Functional dissection of the domains of PUM1&2 identified a conserved amino-terminal region that confers the predominant repressive activity via direct interaction with CNOT. In addition, we show that the mRNA decapping enzyme, DCP2, has an important role in repression by PUM1&2 amino-terminal regions. Our results support a molecular model of repression by human PUM1&2 via direct recruitment of CNOT deadenylation machinery in a decapping-dependent mRNA decay pathway.


Asunto(s)
ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Receptores CCR4/genética , Factores de Transcripción/genética , Transcriptoma , Adenosina Monofosfato , Secuencia de Bases , Sitios de Unión , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , Regulación de la Expresión Génica , Genes Reporteros , Células HCT116 , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Unión Proteica , Estabilidad del ARN , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Receptores CCR4/metabolismo , Factores de Transcripción/metabolismo
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