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1.
Int J Mol Sci ; 22(6)2021 Mar 17.
Artículo en Inglés | MEDLINE | ID: mdl-33802812

RESUMEN

Targeting tumor vasculature through specific endothelial cell markers represents a promising approach for cancer treatment. Here our aim was to construct an antibiotic resistance gene-free plasmid encoding shRNAs to simultaneously target two endothelial cell markers, CD105 and CD146, and to test its functionality and therapeutic potential in vitro when delivered by gene electrotransfer (GET) and combined with irradiation (IR). Functionality of the plasmid was evaluated by determining the silencing of the targeted genes using qRT-PCR. Antiproliferative and antiangiogenic effects were determined by the cytotoxicity assay tube formation assay and wound healing assay in murine endothelial cells 2H-11. The functionality of the plasmid construct was also evaluated in malignant melanoma tumor cell line B16F10. Additionally, potential activation of immune response was measured by induction of DNA sensor STING and proinflammatory cytokines by qRT-PCR in endothelial cells 2H-11. We demonstrated that the plasmid construction was successful and can efficiently silence the expression of the two targeted genes. As a consequence of silencing, reduced migration rate and angiogenic potential was confirmed in 2H-11 endothelial cells. Furthermore, induction of DNA sensor STING and proinflammatory cytokines were determined, which could add to the therapeutic effectiveness when used in vivo. To conclude, we successfully constructed a novel plasmid DNA with two shRNAs, which holds a great promise for further in vivo testing.


Asunto(s)
Antígeno CD146/genética , Electroporación , Endoglina/genética , Silenciador del Gen , Plásmidos/genética , Radiación Ionizante , Transfección , Animales , Muerte Celular , Línea Celular , Citocinas/metabolismo , Células Endoteliales/efectos de la radiación , Proteínas de la Membrana , Ratones , Neovascularización Fisiológica/efectos de la radiación
2.
Int J Mol Sci ; 22(5)2021 Mar 04.
Artículo en Inglés | MEDLINE | ID: mdl-33806461

RESUMEN

The present study aimed to synthesize novel polycationic polymers composed of N-substituted L-2,3-diaminopropionic acid residues (DAPEGs) and investigate their cell permeability, cytotoxicity, and DNA-binding ability. The most efficient cell membrane-penetrating compounds (O2Oc-Dap(GO2)n-O2Oc-NH2, where n = 4, 6, and 8) showed dsDNA binding with a binding constant in the micromolar range (0.3, 3.4, and 0.19 µM, respectively) and were not cytotoxic to HB2 and MDA-MB-231 cells. Selected compounds used in the transfection of a GFP plasmid showed high transfection efficacy and minimal cytotoxicity. Their interaction with plasmid DNA and the increasing length of the main chain of tested compounds strongly influenced the organization and shape of the flower-like nanostructures formed, which were unique for 5/6-FAM-O2Oc-[Dap(GO2)]8-O2Oc-NH2 and typical for large proteins.


Asunto(s)
Permeabilidad de la Membrana Celular/fisiología , Ácidos Nucleicos/metabolismo , Polímeros/farmacología , beta-Alanina/análogos & derivados , Línea Celular , Línea Celular Tumoral , Humanos , Nanoestructuras/química , Plásmidos/metabolismo , Transfección/métodos , beta-Alanina/farmacología
3.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 29(2): 322-327, 2021 Apr.
Artículo en Chino | MEDLINE | ID: mdl-33812394

RESUMEN

OBJECTIVE: To construct an acute myeloid leukemia cell line stably expressing CD123-CLL1 so as to provide an "in vitro" model for studying the role of CD123 and CLL-1 in leukemia and the treatment targeting CD123 and CLL-1. METHODS: The recombinant plasmid of lentivirus was constructed by synthesizing CD123 and CLL-1 sequences and PCR homologous recombination. The lentivirus vector was packaged by three-plasmid packaging system. After collecting the supernatant of lentivirus, the virus titer was determined by quantitative PCR. K562 leukemia cells were collected and transtected with virus supernatant. Leukemia cell line stably expressing the target gene were screened by purinomycin. The expression levels of CD123 and CLL-1 were detected by RT-PCR and flow cytometry. RESULTS: The lentiviral vector was successfully constructed, and identified by agarose gel electrophoresis and gene sequencing, then the virus titer of the supernatant was up to 5.81×108 after quantitative PCR assay. The K562 leukemia cell line obtained positive expression cells after being infected by puromycin. The high expression of CD123 and CLL-1 was confirmed by RT-PCR, while the significantly high expression of CD123 and CLL-1 was confirmed by flow cytometry. CONCLUSION: Lentiviral vector expressing CD123-CLL1 has been successfully constructed, and K562 leukemia cell line stably expressing CD123 and CLL-1 has been successfully obtained.


Asunto(s)
Subunidad alfa del Receptor de Interleucina-3 , Leucemia Linfocítica Crónica de Células B , Línea Celular Tumoral , Vectores Genéticos , Humanos , Células K562 , Lentivirus/genética , Leucemia Linfocítica Crónica de Células B/genética , Plásmidos , Transfección
4.
World J Surg Oncol ; 19(1): 131, 2021 Apr 21.
Artículo en Inglés | MEDLINE | ID: mdl-33882945

RESUMEN

BACKGROUND: Radiotherapy is a main therapeutic method for cancers, including colon cancer. In the current study, we aim to explore the effects of circular RNA (circRNA) circ_0055625 in the progression and radiosensitivity of colon cancer and the underlying mechanism. METHODS: The expression of circ_0055625 and musashi homolog 1 (MSI1) mRNA was detected by quantitative real-time polymerase chain reaction (qRT-PCR). MSI1 protein expression was determined by Western blot. Cell proliferation was assessed by cell counting kit-8 (CCK-8) and colony formation assays. Cell survival fraction, apoptosis, and invasion were investigated by colony formation assay, flow cytometry analysis, and transwell invasion assay, respectively. Cell migration was detected by wound-healing and transwell migration assays. The binding relationship between microRNA-338-3p (miR-338-3p) and circ_0055625 or MSI1 was predicted by online databases and identified by Dual-Luciferase Reporter Assay. The effects of circ_0055625 silencing on the tumor formation and radiosensitivity of colon cancer in vivo were explored by in vivo tumor formation assay. RESULTS: Circ_0055625 and MSI1 were upregulated in colon cancer tissues and cells relative to control groups. Radiation treatment apparently increased the expression of circ_0055625 and MSI1 in colon cancer cells. Circ_0055625 knockdown or MSI1 silencing repressed cell proliferation, migration, and invasion and promoted cell apoptosis and radiosensitivity in colon cancer. Also, circ_0055625 silencing-mediated effects were attenuated by MSI1 overexpression. Additionally, circ_0055625 silencing reduced MSI1 expression, which could be attenuated by miR-338-3p inhibitor. Mechanically, circ_0055625 acted as a sponge for miR-338-3p to regulate MSI1. Furthermore, circ_0055625 knockdown hindered tumor growth and improved radiosensitivity in vivo. CONCLUSION: Circ_0055625 repression inhibited the progression and radioresistance of colon cancer by downregulating MSI1 through sponging miR-338-3p. This result might provide a theoretical basis for improving the therapy of colon cancer with radiation.


Asunto(s)
Neoplasias del Colon , MicroARNs/genética , Proteínas del Tejido Nervioso/genética , ARN Circular/genética , Proteínas de Unión al ARN/genética , Carcinogénesis/genética , Línea Celular Tumoral , Neoplasias del Colon/genética , Neoplasias del Colon/radioterapia , Regulación Neoplásica de la Expresión Génica/genética , Técnicas de Inactivación de Genes , Silenciador del Gen , Humanos , Proteínas del Tejido Nervioso/biosíntesis , Pronóstico , Proteínas de Unión al ARN/biosíntesis , Tolerancia a Radiación/genética , Tolerancia a Radiación/efectos de la radiación , Transfección
5.
Cells ; 10(4)2021 03 28.
Artículo en Inglés | MEDLINE | ID: mdl-33800686

RESUMEN

Parkinson's disease (PD) is the most common neurodegenerative movement disorder, characterized by progressive loss of dopaminergic neurons in the substantia nigra, intraneuronal deposition of misfolded proteins known as Lewy bodies, and chronic neuroinflammation. PD can arise from monogenic mutations, but in most cases, the etiology is unclear. Viral infection is gaining increasing attentions as a trigger of PD. In this study, we investigated whether the PD-causative 620 aspartate (D) to asparagine (N) mutation in the vacuolar protein sorting 35 ortholog (Vps35) precipitated herpes simplex virus (HSV) infection. We observed that ectopic expression of Vps35 significantly reduced the proliferation and release of HSV-1 virions; the D620N mutation rendered Vps35 a partial loss of such inhibitory effects. Tetherin is a host cell protein capable of restricting the spread of encapsulated viruses including HSV-1 and SARS-Cov-2, both of which are implicated in the development of parkinsonism. Compared with cells overexpressing wildtype Vps35, cells expressing mutant Vps35 with D620N had less Tetherin on cell surfaces. Real-time and static cell imaging revealed that Tetherin recycled through Vps35-positive endosomes. Expression of Vps35 with D620N reduced endosomal dynamics and frequency of motile Tetherin-containing vesicles, a sign of defective production of recycling carriers. Our study suggests that the D620N mutation in Vps35 hinders Tetherin trafficking to cell surfaces and facilitates virus spread.


Asunto(s)
Antígeno 2 del Estroma de la Médula Ósea/metabolismo , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/virología , Simplexvirus/metabolismo , Proteínas de Transporte Vesicular/metabolismo , /virología , Línea Celular Tumoral , Endosomas/metabolismo , Humanos , Mutación , Enfermedad de Parkinson/genética , Transporte de Proteínas/genética , /metabolismo , Simplexvirus/patogenicidad , Transfección , Proteínas de Transporte Vesicular/genética , Replicación Viral/genética
6.
Int J Mol Sci ; 22(5)2021 Mar 04.
Artículo en Inglés | MEDLINE | ID: mdl-33806448

RESUMEN

Lambda interferons mediate antiviral immunity by inducing interferon-stimulated genes (ISGs) in epithelial tissues. A common variant rs368234815TT/∆G creating functional gene from an IFNL4 pseudogene is associated with the expression of major ISGs in the liver but impaired clearance of hepatitis C. To explain this, we compared Halo-tagged and non-tagged IFNL3 and IFNL4 signaling in liver-derived cell lines. Transfection with non-tagged IFNL3, non-tagged IFNL4 and Halo-tagged IFNL4 led to a similar degree of JAK-STAT activation and ISG induction; however, the response to transfection with Halo-tagged IFNL3 was lower and delayed. Transfection with non-tagged IFNL3 or IFNL4 induced no transcriptome change in the cells lacking either IL10R2 or IFNLR1 receptor subunits. Cytosolic overexpression of signal peptide-lacking IFNL3 or IFNL4 in wild type cells did not interfere with JAK-STAT signaling triggered by interferons in the medium. Finally, expression profile changes induced by transfection with non-tagged IFNL3 and IFNL4 were highly similar. These data do not support the hypothesis about IFNL4-specific non-canonical signaling and point out that functional studies conducted with tagged interferons should be interpreted with caution.


Asunto(s)
Hepatocitos/inmunología , Hepatocitos/metabolismo , Interferones/genética , Interferones/metabolismo , Interleucinas/genética , Interleucinas/metabolismo , Línea Celular , Expresión Génica , Técnicas de Inactivación de Genes , Células Hep G2 , Humanos , Factores Reguladores del Interferón/genética , Factores Reguladores del Interferón/metabolismo , Interferones/deficiencia , Subunidad beta del Receptor de Interleucina-10/deficiencia , Subunidad beta del Receptor de Interleucina-10/genética , Subunidad beta del Receptor de Interleucina-10/metabolismo , Interleucinas/deficiencia , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Interferón/deficiencia , Receptores de Interferón/genética , Receptores de Interferón/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal , Transfección
7.
Braz J Med Biol Res ; 54(5): e10743, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33825778

RESUMEN

Amphiphilic copolymers have a wide variety of medical and biotechnological applications, including DNA transfection in eukaryotic cells. Still, no polymer-primed transfection of prokaryotic cells has been described. The reversible addition-fragmentation chain transfer (RAFT) polymer synthesis technique and the reversible deactivation radical polymerization variants allow the design of polymers with well-controlled molar mass, morphology, and hydrophilicity/hydrophobicity ratios. RAFT was used to synthesize two amphiphilic copolymers containing different ratios of the amphiphilic poly[2-(dimethyl-amino) ethyl methacrylate] and the hydrophobic poly [methyl methacrylate]. These copolymers bound to pUC-19 DNA and successfully transfected non-competent Escherichia coli DH5α, with transformation efficiency in the range of 103 colony-forming units per µg of plasmid DNA. These results demonstrate prokaryote transformation using polymers with controlled amphiphilic/hydrophobic ratios.


Asunto(s)
ADN , Polímeros , Bacterias , Cationes , ADN/genética , Transfección
8.
Nat Commun ; 12(1): 2121, 2021 04 09.
Artículo en Inglés | MEDLINE | ID: mdl-33837189

RESUMEN

Prime editors (PEs) mediate genome modification without utilizing double-stranded DNA breaks or exogenous donor DNA as a template. PEs facilitate nucleotide substitutions or local insertions or deletions within the genome based on the template sequence encoded within the prime editing guide RNA (pegRNA). However, the efficacy of prime editing in adult mice has not been established. Here we report an NLS-optimized SpCas9-based prime editor that improves genome editing efficiency in both fluorescent reporter cells and at endogenous loci in cultured cell lines. Using this genome modification system, we could also seed tumor formation through somatic cell editing in the adult mouse. Finally, we successfully utilize dual adeno-associated virus (AAVs) for the delivery of a split-intein prime editor and demonstrate that this system enables the correction of a pathogenic mutation in the mouse liver. Our findings further establish the broad potential of this genome editing technology for the directed installation of sequence modifications in vivo, with important implications for disease modeling and correction.


Asunto(s)
Carcinogénesis/genética , Edición Génica/métodos , Neoplasias/genética , ARN Guia/genética , Alelos , Animales , Sistemas CRISPR-Cas/genética , Dependovirus/genética , Modelos Animales de Enfermedad , Células HEK293 , Células HeLa , Humanos , Ratones , Neoplasias/patología , Transfección
9.
Methods Mol Biol ; 2265: 621-634, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33704743

RESUMEN

RNA interference (RNAi) is a posttranscriptional regulatory mechanism that employs siRNA. It typically results in the degradation of a target mRNA that encodes a particular protein. Treatment with siRNA therapeutics requires the use of an effective drug delivery system to assist in delivering these therapeutics into the cytoplasm of the transfected cells. Here we describe the transfection of melanoma cancer cells with siRNA using cationic niosome nanoparticles as a delivery system. The method of niosome preparation is first introduced and is followed by complex formation with siRNA and the transfection method.


Asunto(s)
Melanoma , Nanopartículas , ARN Interferente Pequeño , Transfección , Humanos , Liposomas , Melanoma/genética , Melanoma/metabolismo , Melanoma/patología , Melanoma/terapia , Nanopartículas/química , Nanopartículas/uso terapéutico , ARN Interferente Pequeño/química , ARN Interferente Pequeño/farmacología
10.
Yi Chuan ; 43(3): 280-288, 2021 Mar 16.
Artículo en Inglés | MEDLINE | ID: mdl-33724212

RESUMEN

To improve the transfection efficiency of chicken primordial germ cells (PGCs), the present study evaluated the plasmid dosage and cell number on the efficiencies of three transfection reagents (Lipofectamine 2000, 3000 and LTX & Plus Reagent). PGCs was isolated from embryonic gonads of Huiyang bearded chicken. After 60 days of culture in vitro, the cells were transfected by using Lipofectamine transfection reagents with piggyBac vectors coding for the green fluorescence protein (GFP). PGCs were passaged in culture and fluorescent cells were screened and selected by flow cytometry at three days after transfection. At three weeks post transfection, about 2000 cells were injected into the stage 16 Hamburger and Hamilton (HH) embryos and incubated until stage 30 HH. The results showed that Lipofectamine 3000 was the best for transfection of PGCs. The highest transfection efficiency of PGCs could be achieved with a combination of 3 µg plasmid, 4 µL Lipofectamine 3000 transfection reagent and 0.5×10 4PGCs cells. Flow cytometry analysis showed a 23.4% efficiency of stable transfection of PGCs using Lipofectamine 3000 with piggyBac vector, which was improved 2 times or more over current commonly used methods. After reinjecting PGCs into recipient chicken embryos, GFP-positive cells were observed in the gonads of the recipient chicken embryo by fluorescence microscopy. The study comprehensively evaluated the factors of transfection reagents, plasmid dosage and cell number to optimize the transfection of PGCs, thereby providing a foundation for the efficient preparation of transgenic and gene-edited chickens.


Asunto(s)
Pollos , Células Germinativas , Animales , Animales Modificados Genéticamente , Embrión de Pollo , Pollos/genética , Gónadas , Transfección
11.
Methods Mol Biol ; 2265: 25-46, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33704703

RESUMEN

Recent advances in the treatment of metastatic melanoma have emerged only from advances in our understanding of melanoma development and progression at the cellular and molecular levels. Despite the impact that such advances have made on the clinical management of this cancer over the last decade, additional insights into factors that promote melanoma progression and therapeutic resistance are needed to combat this disease. CRISPR-Cas9 gene editing technology is a powerful tool for studying gene function in a timely and cost-effective manner, enabling the manipulation of specific DNA sequences via a targeted approach. Herein, we describe a protocol for generating functional gene knockouts in melanoma cell lines by CRISPR-Cas9 gene editing, and we present an example application of this protocol for the successful knockout of the Foxc2 transcription factor-encoding gene in the B16-F1 murine melanoma cell line.


Asunto(s)
Edición Génica/métodos , Técnicas de Inactivación de Genes/métodos , Melanoma/genética , Animales , Sistemas CRISPR-Cas , Línea Celular , Factores de Transcripción Forkhead/genética , Vectores Genéticos , Ratones , Transfección
12.
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
13.
Int J Mol Sci ; 22(4)2021 Feb 16.
Artículo en Inglés | MEDLINE | ID: mdl-33669244

RESUMEN

Aurora kinases are serine/threonine kinases required for cell proliferation and are overexpressed in many human cancers. Targeting Aurora kinases has been a therapeutic strategy in cancer treatment. Here, we attempted to identify a deubiquitinase (DUB) that regulates Aurora kinase A (Aurora-A) protein stability and/or kinase activity as a potential cancer therapeutic target. Through pull-down assays with the human DUB library, we identified OTUD6A as an Aurora-A-specific DUB. OTUD6A interacts with Aurora-A through OTU and kinase domains, respectively, and deubiquitinates Aurora-A. Notably, OTUD6A promotes the protein half-life of Aurora-A and activates Aurora-A by increasing phosphorylation at threonine 288 of Aurora-A. From qPCR screening, we identified and validated that the cancer gene CKS2 encoding Cyclin-dependent kinases regulatory subunit 2 is the most upregulated cell cycle regulator when OTUD6A is overexpressed. The results suggest that OTUD6A may serve as a therapeutic target in human cancers.


Asunto(s)
Aurora Quinasa A/genética , Aurora Quinasa A/metabolismo , Enzimas Desubicuitinizantes/genética , Enzimas Desubicuitinizantes/metabolismo , Quinasas CDC2-CDC28/genética , Proteínas de Ciclo Celular/genética , Expresión Génica , Células HEK293 , Semivida , Humanos , Sistemas de Lectura Abierta , Fosforilación , Estabilidad Proteica , Reacción en Cadena en Tiempo Real de la Polimerasa , Transcripción Genética/genética , Transfección
14.
Int J Mol Sci ; 22(4)2021 Feb 16.
Artículo en Inglés | MEDLINE | ID: mdl-33669292

RESUMEN

The ABCG2 gene is a well-established hyperuricemia/gout risk locus encoding a urate transporter that plays a crucial role in renal and intestinal urate excretion. Hitherto, p.Q141K-a common variant of ABCG2 exhibiting approximately one half the cellular function compared to the wild-type-has been reportedly associated with early-onset gout in some populations. However, compared with adult-onset gout, little clinical information is available regarding the association of other uricemia-associated genetic variations with early-onset gout; the latent involvement of ABCG2 in the development of this disease requires further evidence. We describe a representative case of familial pediatric-onset hyperuricemia and early-onset gout associated with a dysfunctional ABCG2, i.e., a clinical history of three generations of one Czech family with biochemical and molecular genetic findings. Hyperuricemia was defined as serum uric acid (SUA) concentrations 420 µmol/L for men or 360 µmol/L for women and children under 15 years on two measurements, performed at least four weeks apart. The proband was a 12-year-old girl of Roma ethnicity, whose SUA concentrations were 397-405 µmol/L. Sequencing analyses focusing on the coding region of ABCG2 identified two rare mutations-c.393G>T (p.M131I) and c.706C>T (p.R236X). Segregation analysis revealed a plausible link between these mutations and hyperuricemia and the gout phenotype in family relatives. Functional studies revealed that p.M131I and p.R236X were functionally deficient and null, respectively. Our findings illustrate why genetic factors affecting ABCG2 function should be routinely considered in clinical practice as part of a hyperuricemia/gout diagnosis, especially in pediatric-onset patients with a strong family history.


Asunto(s)
Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/genética , Gota/complicaciones , Gota/genética , Hiperuricemia/complicaciones , Hiperuricemia/genética , Proteínas de Neoplasias/genética , Transportadores de Anión Orgánico/genética , Polimorfismo de Nucleótido Simple , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Adulto , Niño , República Checa , Femenino , Predisposición Genética a la Enfermedad , Células HEK293 , Humanos , Hiperuricemia/sangre , Masculino , Mutación , Proteínas de Neoplasias/metabolismo , Transportadores de Anión Orgánico/metabolismo , Linaje , Fenotipo , Transfección , Ácido Úrico/sangre
15.
Int J Mol Sci ; 22(4)2021 Feb 16.
Artículo en Inglés | MEDLINE | ID: mdl-33669407

RESUMEN

La Reunion island in the South West Indian Ocean is now endemic for dengue following the introduction of dengue virus serotype 2 (DENV-2) cosmopolitan-I genotype in 2017. DENV-2 infection causes a wide spectrum of clinical manifestations ranging from flu-like disease to severe dengue. The nonstructural glycoprotein 1 (NS1) has been identified as playing a key role in dengue disease severity. The intracellular NS1 exists as a homodimer, whereas a fraction is driven towards the plasma membrane or released as a soluble hexameric protein. Here, we characterized the NS1 glycoproteins from clinical isolates DES-14 and RUN-18 that were collected during the DENV-2 epidemics in Tanzania in 2014 and La Reunion island in 2018, respectively. In relation to hepatotropism of the DENV, expression of recombinant DES-14 NS1 and RUN-18 NS1 glycoproteins was compared in human hepatoma Huh7 cells. We observed that RUN-18 NS1 was poorly stable in Huh7 cells compared to DES-14 NS1. The instability of RUN-18 NS1 leading to a low level of NS1 secretion mostly relates to lysine residues on positions 272 and 324. Our data raise the issue of the consequences of a defect in NS1 stability in human hepatocytes in relation to the major role of NS1 in the pathogenesis of the DENV-2 infection.


Asunto(s)
Virus del Dengue/metabolismo , Dengue/epidemiología , Dengue/metabolismo , Epidemias , Genotipo , Lisina/química , Proteínas no Estructurales Virales/química , Sustitución de Aminoácidos , Antígenos Virales/química , Antígenos Virales/genética , Línea Celular Tumoral , Dengue/virología , Células HEK293 , Hepatocitos/metabolismo , Hepatocitos/virología , Humanos , Multimerización de Proteína , Estabilidad Proteica , Proteínas Recombinantes/química , Reunión/epidemiología , Serogrupo , Tanzanía/epidemiología , Transfección , Proteínas no Estructurales Virales/genética
16.
J Vis Exp ; (168)2021 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-33645580

RESUMEN

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells leave their quiescent state and activate the myogenic program ultimately leading to the repair of damaged tissue concomitant with the replenishment of the muscle stem cell pool. Various factors influence muscle stem cell activity, among them intrinsic stimuli but also signals from the direct muscle stem cell environment, the stem cell niche. The isolation and culture of single myofibers with their associated muscle stem cells preserves most of the interaction of the stem cell with its niche and is, therefore, the closest possibility to study muscle stem cell functionality ex vivo. Here, a protocol for the isolation, culture, siRNA transfection and immunostaining of muscle stem cells on their respective myofibers from mouse EDL (extensor digitorum longus) muscles is provided. The experimental conditions outlined here allow the study and manipulation of muscle stem cells ex vivo including investigation of myogenic activity without the inherent need for in vivo animal experiments.


Asunto(s)
Células Madre Adultas/citología , Técnicas de Cultivo de Célula/métodos , Fibras Musculares Esqueléticas/citología , Células Madre/citología , Animales , Células Cultivadas , Colagenasas/metabolismo , Ratones Endogámicos C57BL , Desarrollo de Músculos , ARN Interferente Pequeño/metabolismo , Regeneración , Fijación del Tejido , Transfección
17.
BMC Cancer ; 21(1): 310, 2021 Mar 24.
Artículo en Inglés | MEDLINE | ID: mdl-33761896

RESUMEN

BACKGROUND: Chromosomal inversions involving anaplastic lymphoma kinase (ALK) and echinoderm microtubule associated protein like 4 (EML4) generate a fusion protein EML4-ALK in non-small cell lung cancer (NSCLC). The understanding of EML4-ALK function can be improved by a functional study using normal human cells. METHODS: Here we for the first time conduct such study to examine the effects of EML4-ALK on cell proliferation, cellular senescence, DNA damage, gene expression profiles and transformed phenotypes. RESULTS: The lentiviral expression of EML4-ALK in mortal, normal human fibroblasts caused, through its constitutive ALK kinase activity, an early induction of cellular senescence with accumulated DNA damage, upregulation of p16INK4A and p21WAF1, and senescence-associated ß-galactosidase (SA-ß-gal) activity. In contrast, when EML4-ALK was expressed in normal human fibroblasts transduced with telomerase reverse transcriptase (hTERT), which is activated in the vast majority of NSCLC, the cells showed accelerated proliferation and acquired anchorage-independent growth ability in soft-agar medium, without accumulated DNA damage, chromosome aberration, nor p53 mutation. EML4-ALK induced the phosphorylation of STAT3 in both mortal and hTERT-transduced cells, but RNA sequencing analysis suggested that the different signaling pathways contributed to the different phenotypic outcomes in these cells. While EML4-ALK also induced anchorage-independent growth in hTERT-immortalized human bronchial epithelial cells in vitro, the expression of EML4-ALK alone did not cause detectable in vivo tumorigenicity in immunodeficient mice. CONCLUSIONS: Our data indicate that the expression of hTERT is critical for EML4-ALK to manifest its in vitro transforming activity in human cells. This study provides the isogenic pairs of human cells with and without EML4-ALK expression.


Asunto(s)
Carcinogénesis/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Neoplasias Pulmonares/genética , Proteínas de Fusión Oncogénica/metabolismo , Telomerasa/metabolismo , Animales , Carcinogénesis/patología , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular , Proliferación Celular/genética , Senescencia Celular/genética , Daño del ADN , Modelos Animales de Enfermedad , Células Epiteliales , Femenino , Fibroblastos , Regulación Neoplásica de la Expresión Génica , Vectores Genéticos/genética , Humanos , Lentivirus/genética , Neoplasias Pulmonares/patología , Ratones , Proteínas de Fusión Oncogénica/genética , RNA-Seq , Telomerasa/genética , Homeostasis del Telómero/genética , Transfección
18.
Methods Mol Biol ; 2235: 139-153, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33576975

RESUMEN

MicroRNAs (miRNAs) are expressed in all cell types, including pericytes, and play essential roles in vascular development, homeostasis, and disease. Manipulation of pericytes with miRNA mimics and inhibitors represents an essential tool to study the role of pericytes in vascular development and regeneration and to better understand the therapeutic potential of miRNA manipulation in pericytes. Here we describe methods for manipulating pericyte function by using miRNA mimics and inhibitors. We also describe methods to assess pericyte function (proliferation and migration) after manipulation with miRNAs and explain how miRNA gene targets can be identified and validated in pericytes after manipulation with miRNA.


Asunto(s)
Clonación Molecular/métodos , MicroARNs/genética , Pericitos/metabolismo , Animales , Regulación de la Expresión Génica/genética , Humanos , MicroARNs/fisiología , Pericitos/fisiología , Transfección/métodos , Transformación Genética/genética
19.
Methods Mol Biol ; 2244: 115-132, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33555585

RESUMEN

Human fibroblasts represent the most extensively used cell type for the investigation of lytic human cytomegalovirus (HCMV) replication. However, analyzing the function of specific proteins during infection can be challenging since primary cells are difficult to transfect. An alternative approach is the use of lentiviral transduction with vectors for stable or inducible shRNA expression. This approach provides a versatile tool to study the role of host cell factors during HCMV infection. The essential steps to achieve an efficient target protein knockdown are shRNA design, cloning, generation of transgenic lentiviral particles, and, finally, transduction of the cells. However, these steps are highly dependent on the selected vector system. Here we focus on two different vector systems and describe how to successfully generate stable and inducible knockdown fibroblasts. Additionally, we demonstrate different methods to validate the knockdown of the target protein.


Asunto(s)
Citomegalovirus/metabolismo , Técnicas de Silenciamiento del Gen/métodos , Cultivo Primario de Células/métodos , Línea Celular , Infecciones por Citomegalovirus/virología , Fibroblastos/citología , Fibroblastos/metabolismo , Vectores Genéticos/genética , Humanos , ARN Interferente Pequeño/genética , Transfección/métodos , Proteínas Virales , Replicación Viral/fisiología
20.
Methods Mol Biol ; 2244: 133-158, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33555586

RESUMEN

To fully understand the function of cytomegalovirus (CMV) genes, it is imperative that they are studied in the context of infection. Therefore, the targeted deletion of individual viral genes and the comparison of these loss-of-function viral mutants to the wild-type virus allow for the identification of the relevance and role for a particular gene in the viral replication cycle. Targeted CMV mutagenesis has made huge advances over the past 20 years. The cloning of CMV genomes into Escherichia coli as bacterial artificial chromosomes (BAC) allows for not only quick and efficient deletion of viral genomic regions, individual genes, or single-nucleotide exchanges in the viral genome but also the insertion of heterologous genetic sequences for gain-of-function approaches. The conceptual advantage of this strategy is that it overcomes the restrictions of recombinant technologies in cell culture systems. Namely, recombination in infected cells occurs only in a few clones, and their selection is not possible if the targeted genes are relevant for virus replication and are not able to compete for growth against the unrecombined parental viruses. On the other hand, BAC mutagenesis enables the selection for antibiotic resistance in E. coli, providing selective growth advantage to the recombined genomes and thus clonal selection of viruses with even extremely poor fitness. Here we describe the methods used for the generation of a CMV BAC, targeted mutagenesis of BAC clones, and transfection of human cells with CMV BAC DNA in order to reconstitute the viral infection process.


Asunto(s)
Cromosomas Artificiales Bacterianos/genética , Clonación Molecular/métodos , Citomegalovirus/genética , Células Cultivadas , Escherichia coli/genética , Genes Virales/genética , Genoma Viral/genética , Humanos , Mutagénesis/genética , Transfección/métodos , Replicación Viral/genética
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