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1.
Int J Mol Sci ; 22(16)2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34445497

RESUMO

Membrane proteins responsible for transporting magnetic resonance (MR) and fluorescent contrast agents are of particular importance because they are potential reporter proteins in noninvasive molecular imaging. Gadobenate dimeglumine (Gd-BOPTA), a liver-specific MR contrast agent, has been used globally for more than 10 years. However, the corresponding molecular transportation mechanism has not been validated. We previously reported that the organic anion transporting polypeptide (OATP) 1B3 has an uptake capability for both MR agents (Gd-EOB-DTPA) and indocyanine green (ICG), a clinically available near-infrared (NIR) fluorescent dye. This study further evaluated OATP1B1, another polypeptide of the OATP family, to determine its reporter capability. In the OATP1B1 transfected 293T transient expression model, both Gd-BOPTA and Gd-EOB-DTPA uptake were confirmed through 1.5 T MR imaging. In the constant OAPT1B1 and OATP1B3 expression model in the HT-1080 cell line, both HT-1080-OAPT1B1 and HT-1080-OATP1B3 were observed to ingest Gd-BOPTA and Gd-EOB-DTPA. Lastly, we validated the ICG uptake capability of both OATP1B1 and OATP1B3. OAPT1B3 exhibited a superior ICG uptake capability to that of OAPT1B1. We conclude that OATP1B1 is a potential reporter for dual MR and NIR fluorescent molecular imaging, especially in conjunction with Gd-BOPTA.


Assuntos
Gadolínio DTPA/química , Transportador 1 de Ânion Orgânico Específico do Fígado/metabolismo , Meglumina/análogos & derivados , Imagem Óptica/métodos , Compostos Organometálicos/química , Genes Reporter , Células HEK293 , Humanos , Transportador 1 de Ânion Orgânico Específico do Fígado/química , Transportador 1 de Ânion Orgânico Específico do Fígado/genética , Imageamento por Ressonância Magnética , Meglumina/química , Imagem Molecular , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de Soluto/química , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de Soluto/genética , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de Soluto/metabolismo
2.
Bioengineered ; 12(1): 4407-4419, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34436976

RESUMO

Widespread infection due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) has led to a global pandemic. Currently, various approaches are being taken up to develop vaccines and therapeutics to treat SARS-CoV2 infection. Consequently, the S protein has become an important target protein for developing vaccines and therapeutics against SARS-CoV2. However, the highly infective nature of SARS-CoV2 restricts experimentation with the virus to highly secure BSL3 facilities. The availability of fusion-enabled, nonreplicating, and nonbiohazardous mimics of SARS-CoV2 virus fusion, containing the viral S or S and M protein in their native conformation on mammalian cells, can serve as a useful substitute for studying viral fusion for testing various inhibitors of viral fusion. This would avoid the use of the BSL3 facility for fusion studies required to develop therapeutics. In the present study, we have developed SARS-CoV2 virus fusion mimics (SCFMs) using mammalian cells transfected with constructs coding for S or S and M protein. The fusogenic property of the mimic(s) and their interaction with the functional human ACE2 receptors was confirmed experimentally. We have also shown that such mimics can easily be used in an inhibition assay. These mimic(s) can be easily prepared on a large scale, and such SCFMs can serve as an invaluable resource for viral fusion inhibition assays and in vitro screening of antiviral agents, which can be shared/handled between labs/facilities without worrying about any biohazard while working under routine laboratory conditions, avoiding the use of BSL3 laboratory.Abbreviations :SCFM: SARS-CoV2 Virus Fusion Mimic; ACE2: Angiotensin-Converting Enzyme 2; hACE2: Human Angiotensin-Converting enzyme 2; MEF: Mouse Embryonic Fibroblasts; HBSS: Hanks Balanced Salt Solution; FBS: Fetal Bovine Serum.


Assuntos
Anticorpos Neutralizantes/farmacologia , Contenção de Riscos Biológicos/métodos , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Proteínas da Matriz Viral/antagonistas & inibidores , Internalização do Vírus/efeitos dos fármacos , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Chlorocebus aethiops , Embrião de Mamíferos , Fibroblastos/efeitos dos fármacos , Fibroblastos/virologia , Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Células MCF-7 , Camundongos , Mimetismo Molecular , Plasmídeos/química , Plasmídeos/metabolismo , Cultura Primária de Células , Ligação Proteica , Receptores Virais/genética , Receptores Virais/metabolismo , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Transfecção , Células Vero , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismo
3.
Int J Mol Sci ; 22(12)2021 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-34208268

RESUMO

Euglena gracilis (E. gracilis) is an attractive organism due to its evolutionary history and substantial potential to produce biochemicals of commercial importance. This study describes the establishment of an optimized protocol for the genetic transformation of E. gracilis mediated by Agrobacterium (A. tumefaciens). E. gracilis was found to be highly sensitive to hygromycin and zeocin, thus offering a set of resistance marker genes for the selection of transformants. A. tumefaciens-mediated transformation (ATMT) yielded hygromycin-resistant cells. However, hygromycin-resistant cells hosting the gus gene (encoding ß-glucuronidase (GUS)) were found to be GUS-negative, indicating that the gus gene had explicitly been silenced. To circumvent transgene silencing, GUS was expressed from the nuclear genome as transcriptional fusions with the hygromycin resistance gene (hptII) (encoding hygromycin phosphotransferase II) with the foot and mouth disease virus (FMDV)-derived 2A self-cleaving sequence placed between the coding sequences. ATMT of Euglena with the hptII-2A-gus gene yielded hygromycin-resistant, GUS-positive cells. The transformation was verified by PCR amplification of the T-DNA region genes, determination of GUS activity, and indirect immunofluorescence assays. Cocultivation factors optimization revealed that a higher number of transformants was obtained when A. tumefaciens LBA4404 (A600 = 1.0) and E. gracilis (A750 = 2.0) cultures were cocultured for 48 h at 19 °C in an organic medium (pH 6.5) containing 50 µM acetosyringone. Transformation efficiency of 8.26 ± 4.9% was achieved under the optimized cocultivation parameters. The molecular toolkits and method presented here can be used to bioengineer E. gracilis for producing high-value products and fundamental studies.


Assuntos
Agrobacterium tumefaciens/metabolismo , Biotecnologia , Euglena gracilis/genética , Microalgas/genética , Técnicas de Transferência Nuclear , Transformação Genética , Agrobacterium tumefaciens/efeitos dos fármacos , Antibacterianos/farmacologia , Cinamatos/farmacologia , Células Clonais , DNA Bacteriano/genética , Euglena gracilis/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Genes Reporter , Higromicina B/análogos & derivados , Higromicina B/farmacologia , Microalgas/efeitos dos fármacos , Mutagênese Insercional/genética , Transformação Genética/efeitos dos fármacos , Transgenes
4.
Int J Mol Sci ; 22(13)2021 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-34203261

RESUMO

A group of clinically approved cancer therapeutic tyrosine kinase inhibitors was screened to test their effects on the expression of angiotensin-converting enzyme 2 (ACE2), the cell surface receptor for SARS-CoV-2. Here, we show that the receptor tyrosine kinase inhibitor imatinib (also known as STI571, Gleevec) can inhibit the expression of the endogenous ACE2 gene at both the transcript and protein levels. Treatment with imatinib resulted in inhibition of cell entry of the viral pseudoparticles (Vpps) in cell culture. In FVB mice orally fed imatinib, tissue expression of ACE2 was reduced, specifically in the lungs and renal tubules, but not in the parenchyma of other organs such as the heart and intestine. Our finding suggests that receptor tyrosine kinases play a role in COVID-19 infection and can be therapeutic targets with combined treatments of the best conventional care of COVID-19.


Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , Regulação para Baixo/efeitos dos fármacos , Mesilato de Imatinib/farmacologia , SARS-CoV-2/fisiologia , Internalização do Vírus/efeitos dos fármacos , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/genética , Animais , COVID-19/patologia , COVID-19/virologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Feminino , Genes Reporter , Humanos , Camundongos , Regiões Promotoras Genéticas , SARS-CoV-2/isolamento & purificação
5.
Cancer Immunol Immunother ; 70(9): 2701-2719, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34244816

RESUMO

Recombinant agonists that activate co-stimulatory and cytokine receptors have shown limited clinical anticancer utility, potentially due to narrow therapeutic windows, the need for coordinated activation of co-stimulatory and cytokine pathways and the failure of agonistic antibodies to recapitulate signaling by endogenous ligands. RTX-240 is a genetically engineered red blood cell expressing 4-1BBL and IL-15/IL-15Rα fusion (IL-15TP). RTX-240 is designed to potently and simultaneously stimulate the 4-1BB and IL-15 pathways, thereby activating and expanding T cells and NK cells, while potentially offering an improved safety profile through restricted biodistribution. We assessed the ability of RTX-240 to expand and activate T cells and NK cells and evaluated the in vivo efficacy, pharmacodynamics and tolerability using murine models. Treatment of PBMCs with RTX-240 induced T cell and NK cell activation and proliferation. In vivo studies using mRBC-240, a mouse surrogate for RTX-240, revealed biodistribution predominantly to the red pulp of the spleen, leading to CD8 + T cell and NK cell expansion. mRBC-240 was efficacious in a B16-F10 melanoma model and led to increased NK cell infiltration into the lungs. mRBC-240 significantly inhibited CT26 tumor growth, in association with an increase in tumor-infiltrating proliferating and cytotoxic CD8 + T cells. mRBC-240 was tolerated and showed no evidence of hepatic injury at the highest feasible dose, compared with a 4-1BB agonistic antibody. RTX-240 promotes T cell and NK cell activity in preclinical models and shows efficacy and an improved safety profile. Based on these data, RTX-240 is now being evaluated in a clinical trial.


Assuntos
Ligante 4-1BB/genética , Terapia Baseada em Transplante de Células e Tecidos , Eritrócitos/metabolismo , Expressão Gênica , Terapia Genética , Interleucina-15/genética , Ligante 4-1BB/metabolismo , Animais , Terapia Baseada em Transplante de Células e Tecidos/métodos , Células Precursoras Eritroides/metabolismo , Feminino , Citometria de Fluxo , Genes Reporter , Engenharia Genética , Terapia Genética/métodos , Humanos , Interleucina-15/metabolismo , Camundongos , Modelos Animais , Ligação Proteica , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Methods Mol Biol ; 2350: 229-237, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34331288

RESUMO

The recent development of the bright luciferase NanoLuc (Nluc) has greatly improved the sensitivity of bioluminescence imaging, enabling real-time cellular imaging with high spatial resolution. However, the limited color variants of Nluc have restricted its wider application to multicolor imaging of biological phenomena. To address this issue, we developed five new spectral variants of the bright bioluminescent protein with emissions across the visible spectrum. In this chapter, we describe the following two protocols for single-cell bioluminescence imaging: (a) multicolor bioluminescence imaging of subcellular structures and (b) multicolor calcium imaging in single living cells.


Assuntos
Cálcio/metabolismo , Medições Luminescentes/métodos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Imagem Molecular/métodos , Análise de Célula Única/métodos , Técnicas de Transferência de Energia por Ressonância de Bioluminescência , Linhagem Celular , Expressão Gênica , Genes Reporter , Humanos
7.
Methods Mol Biol ; 2341: 37-44, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34264459

RESUMO

The lacZ gene and corresponding ß-galactosidase enzyme has been a mainstay for bacterial reporter systems for decades. We have used this versatile reporter to analyze expression profiles from strains grown both on solid media and from broth culture. The standard broth protocol can also be adapted for a 96-well plate to allow high-throughput screening of promoter reporter constructs under a variety of conditions. Furthermore, codon-optimization of the E. coli lacZ gene has greatly improved activity levels of ß-galactosidase in S. aureus, facilitating improved sensitivity for screening assays, detection of low-activity promoters, and use of small sample volumes. In this chapter, details are provided for both standard and high-throughput quantitative assays that we have routinely used for S. aureus transcriptional profiling.


Assuntos
Toxinas Bacterianas/genética , Escherichia coli/enzimologia , Proteínas Hemolisinas/genética , Ensaios de Triagem em Larga Escala/métodos , Staphylococcus aureus/genética , beta-Galactosidase/genética , Proteínas de Bactérias/genética , Composição de Bases , Códon , Escherichia coli/genética , Expressão Gênica , Genes Reporter , Óperon Lac , Regiões Promotoras Genéticas
8.
Int J Mol Sci ; 22(13)2021 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-34203212

RESUMO

Firefly luciferase is susceptible to inhibition and stabilization by compounds under investigation for biological activity and toxicity. This can lead to false-positive results in in vitro cell-based assays. However, firefly luciferase remains one of the most commonly used reporter genes. Here, we evaluated isoflavonoids for inhibition of firefly luciferase. These natural compounds are often studied using luciferase reporter-gene assays. We used a quantitative structure-activity relationship (QSAR) model to compare the results of in silico predictions with a newly developed in vitro assay that enables concomitant detection of inhibition of firefly and Renilla luciferases. The QSAR model predicted a moderate to high likelihood of firefly luciferase inhibition for all of the 11 isoflavonoids investigated, and the in vitro assays confirmed this for seven of them: daidzein, genistein, glycitein, prunetin, biochanin A, calycosin, and formononetin. In contrast, none of the 11 isoflavonoids inhibited Renilla luciferase. Molecular docking calculations indicated that isoflavonoids interact favorably with the D-luciferin binding pocket of firefly luciferase. These data demonstrate the importance of reporter-enzyme inhibition when studying the effects of such compounds and suggest that this in vitro assay can be used to exclude false-positives due to firefly or Renilla luciferase inhibition, and to thus define the most appropriate reporter gene.


Assuntos
Genes Reporter/fisiologia , Isoflavonas/metabolismo , Luciferases de Renilla/metabolismo , Animais , Vaga-Lumes , Genes Reporter/genética , Isoflavonas/química , Luciferases de Renilla/química , Estrutura Secundária de Proteína
9.
Int J Mol Sci ; 22(13)2021 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-34210100

RESUMO

Cas endonuclease-mediated genome editing provides a long-awaited molecular biological approach to the modification of predefined genomic target sequences in living organisms. Although cas9/guide (g)RNA constructs are straightforward to assemble and can be customized to target virtually any site in the plant genome, the implementation of this technology can be cumbersome, especially in species like triticale that are difficult to transform, for which only limited genome information is available and/or which carry comparatively large genomes. To cope with these challenges, we have pre-validated cas9/gRNA constructs (1) by frameshift restitution of a reporter gene co-introduced by ballistic DNA transfer to barley epidermis cells, and (2) via transfection in triticale protoplasts followed by either a T7E1-based cleavage assay or by deep-sequencing of target-specific PCR amplicons. For exemplification, we addressed the triticale ABA 8'-hydroxylase 1 gene, one of the putative determinants of pre-harvest sprouting of grains. We further show that in-del induction frequency in triticalecan beincreased by TREX2 nuclease activity, which holds true for both well- and poorly performing gRNAs. The presented results constitute a sound basis for the targeted induction of heritable modifications in triticale genes.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Edição de Genes/métodos , Proteínas de Plantas/metabolismo , Triticale/metabolismo , Sistemas CRISPR-Cas , Sistema Enzimático do Citocromo P-450/genética , Genes Reporter , Mutação INDEL , Mutagênese Sítio-Dirigida , Proteínas de Plantas/genética , Transfecção , Triticale/genética
10.
Int J Mol Sci ; 22(11)2021 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-34198800

RESUMO

COVID-19 pandemic is caused by betacoronavirus SARS-CoV-2. The genome of this virus is composed of a single strand of RNA with 5' and 3'-UTR flanking a region of protein-coding ORFs closely resembling cells' mRNAs. MicroRNAs are endogenous post-transcriptional regulators that target mRNA to modulate protein expression and mediate cellular functions, including antiviral defense. In the present study, we carried out a bioinformatics screening to search for endogenous human microRNAs targeting the 3'-UTR of SARS-CoV-2. Results from the computational techniques allowed us to identify 10 potential candidates. The capacity of 3 of them, together with hsa-miR-138-5p, to target the SARS-CoV-2 3'-UTR was validated in vitro by gene reporter assays. Available information indicates that two of these microRNAs, namely, hsa-miR-3941 and hsa-miR-138-5p, combine effective targeting of SARS-CoV-2 genome with complementary antiviral or protective effects in the host cells that make them potential candidates for therapeutic treatment of most, if not all, COVID-19 variants known to date. All information obtained while conducting the present analysis is available at Open Science Framework repository.


Assuntos
MicroRNAs/metabolismo , SARS-CoV-2/genética , Regiões 3' não Traduzidas , Sequência de Bases , Sítios de Ligação , COVID-19/genética , COVID-19/patologia , COVID-19/virologia , Linhagem Celular , Genes Reporter , Genoma Viral , Humanos , MicroRNAs/química , Fases de Leitura Aberta , SARS-CoV-2/isolamento & purificação , Alinhamento de Sequência
11.
Int J Mol Sci ; 22(12)2021 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-34207305

RESUMO

The COVID-19 pandemic caused by SARS-CoV-2 coronavirus deeply affected the world community. It gave a strong impetus to the development of not only approaches to diagnostics and therapy, but also fundamental research of the molecular biology of this virus. Fluorescence microscopy is a powerful technology enabling detailed investigation of virus-cell interactions in fixed and live samples with high specificity. While spatial resolution of conventional fluorescence microscopy is not sufficient to resolve all virus-related structures, super-resolution fluorescence microscopy can solve this problem. In this paper, we review the use of fluorescence microscopy to study SARS-CoV-2 and related viruses. The prospects for the application of the recently developed advanced methods of fluorescence labeling and microscopy-which in our opinion can provide important information about the molecular biology of SARS-CoV-2-are discussed.


Assuntos
Microscopia de Fluorescência , SARS-CoV-2/fisiologia , COVID-19/patologia , COVID-19/virologia , Endocitose , Corantes Fluorescentes/química , Genes Reporter , Humanos , RNA Viral/química , RNA Viral/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismo , Internalização do Vírus
12.
Nat Commun ; 12(1): 4586, 2021 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-34321486

RESUMO

Heterogeneous immunoassays such as ELISA have become indispensable in modern bioanalysis, yet translation into point-of-care assays is hindered by their dependence on external calibration and multiple washing and incubation steps. Here, we introduce RAPPID (Ratiometric Plug-and-Play Immunodiagnostics), a mix-and-measure homogeneous immunoassay platform that combines highly specific antibody-based detection with a ratiometric bioluminescent readout. The concept entails analyte-induced complementation of split NanoLuc luciferase fragments, photoconjugated to an antibody sandwich pair via protein G adapters. Introduction of a calibrator luciferase provides a robust ratiometric signal that allows direct in-sample calibration and quantitative measurements in complex media such as blood plasma. We developed RAPPID sensors that allow low-picomolar detection of several protein biomarkers, anti-drug antibodies, therapeutic antibodies, and both SARS-CoV-2 spike protein and anti-SARS-CoV-2 antibodies. With its easy-to-implement standardized workflow, RAPPID provides an attractive, fast, and low-cost alternative to traditional immunoassays, in an academic setting, in clinical laboratories, and for point-of-care applications.


Assuntos
Anticorpos Antivirais/sangue , Teste Sorológico para COVID-19/métodos , COVID-19/diagnóstico , Imunoensaio/normas , Medições Luminescentes/normas , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/sangue , COVID-19/imunologia , COVID-19/virologia , Teste Sorológico para COVID-19/instrumentação , Calibragem , Proteínas de Ligação ao GTP/química , Genes Reporter , Humanos , Imunoconjugados/química , Limite de Detecção , Luciferases/genética , Luciferases/metabolismo , Testes Imediatos , SARS-CoV-2/genética
13.
Int J Mol Sci ; 22(13)2021 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-34202215

RESUMO

Cytokines are important neuroinflammatory modulators in neurodegenerative brain disorders including traumatic brain injury (TBI) and stroke. However, their temporal effects on the physiological properties of microglia and neurons during the recovery period have been unclear. Here, using an ATP-induced cortical injury model, we characterized selective effects of ATP injection compared to needle-control. In the damaged region, the fluorescent intensity of CX3CR1-GFP (+) cells, as well as the cell density, was increased and the maturation of newborn BrdU (+) cells continued until 28 day-post-injection (dpi) of ATP. The excitability and synaptic E/I balance of neurons and the inward and outward membrane currents of microglia were increased at 3 dpi, when expressions of tumor necrosis factor (TNF)-α/interleukin (IL)-1ß and IL-10/IL-4 were also enhanced. These changes of both cells at 3 dpi were mostly decayed at 7 dpi and were suppressed by any of IL-10, IL-4, suramin (P2 receptor inhibitor) and 4-AP (K+ channel blocker). Acute ATP application alone induced only small effects from both naïve neurons and microglial cells in brain slice. However, TNF-α alone effectively increased the excitability of naïve neurons, which was blocked by suramin or 4-AP. TNF-α and IL-1ß increased and decreased membrane currents of naïve microglia, respectively. Our results suggest that ATP and TNF-α dominantly induce the physiological activities of 3 dpi neurons and microglia, and IL-10 effectively suppresses such changes of both activated cells in K+ channel- and P2 receptor-dependent manner, while IL-4 suppresses neurons preferentially.


Assuntos
Lesões Encefálicas Traumáticas/metabolismo , Lesões Encefálicas Traumáticas/patologia , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Potenciais da Membrana , Microglia/fisiologia , Trifosfato de Adenosina/metabolismo , Animais , Lesões Encefálicas Traumáticas/diagnóstico , Lesões Encefálicas Traumáticas/etiologia , Citocinas/metabolismo , Modelos Animais de Doenças , Suscetibilidade a Doenças , Expressão Gênica , Genes Reporter , Camundongos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Antagonistas Purinérgicos/farmacologia
14.
Viruses ; 13(6)2021 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-34204732

RESUMO

The COVID-19 pandemic caused by SARS-CoV-2 has posed a global threat to human lives and economics. One of the best ways to determine protection against the infection is to quantify the neutralizing activity of serum antibodies. Multiple assays have been developed to validate SARS-CoV-2 neutralization; most of them utilized lentiviral or vesicular stomatitis virus-based particles pseudotyped with the spike (S) protein, making them safe and acceptable to work with in many labs. However, these systems are only capable of measuring infection with purified particles. This study has developed a pseudoviral assay with replication-dependent reporter vectors that can accurately quantify the level of infection directly from the virus producing cell to the permissive target cell. Comparative analysis of cell-free and cell-to-cell infection revealed that the neutralizing activity of convalescent sera was more than tenfold lower in cell cocultures than in the cell-free mode of infection. As the pseudoviral system could not properly model the mechanisms of SARS-CoV-2 transmission, similar experiments were performed with replication-competent coronavirus, which detected nearly complete SARS-CoV-2 cell-to-cell infection resistance to neutralization by convalescent sera. These findings suggest that the cell-to-cell mode of SARS-CoV-2 transmission, for which the mechanisms are largely unknown, could be of great importance for treatment and prevention of COVID-19.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Convalescença , Testes de Neutralização/métodos , SARS-CoV-2/imunologia , Genes Reporter/genética , Células HEK293 , Humanos , Testes de Neutralização/normas , SARS-CoV-2/genética
15.
Methods Mol Biol ; 2312: 15-33, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34228282

RESUMO

Synthetic receptors control cell behavior in response to environmental stimuli for applications in basic research and cell therapy. However, the integration of synthetic receptors in unexplored contexts is cumbersome, especially for nonspecialist laboratories. Here, I provide a detailed protocol on how to use receptors of the generalized extracellular molecule sensor (GEMS) platform. GEMS is a modular receptor system that can be adapted to sense molecules of choice by using affinity domains that dimerize in response to the target. GEMS consist of an erythropoietin receptor scaffold that has been mutated to no longer bind to erythropoietin. N-terminal fusions with affinity domains, such as single chain variable fragments (scFvs), that bind to two epitopes on the same target activate the receptor. The intracellular receptor domain can be chosen from several signal transduction domains of single-pass transmembrane receptors to activate endogenous signaling pathways. As of now, GEMS have been used for sensing prostate specific antigen (PSA), the synthetic azo dye RR120, caffeine, nicotine, rapamycin, the SunTag peptide, and a de novo designed protein displaying two viral epitopes. The tested intracellular domains were derived from FGFR1, IL-6RB, and VEGFR2, and were used to drive transgene expression from reporter plasmids responsive to the endogenous transcription factors STAT3, NFAT, NF-κB, and a synthetic transcription factor activated by the MAPK pathway. In this protocol, I focus on transient transfections of HEK293T cells and include several general notes about cell handling. While the described methods are optimized for experiments with GEMS, most of the described techniques are general procedures to set up synthetic biology experiments in mammalian cell culture. I outline how to generate stable cell lines and share tips on how to adapt GEMS for new ligands. The main objective of this protocol is to make the GEMS technology accessible also to nonspecialist laboratories to facilitate the use of synthetic receptors in new research contexts.


Assuntos
Técnicas Biossensoriais , Engenharia Celular , Engenharia de Proteínas , Receptores Artificiais/genética , Receptores da Eritropoetina/genética , Anticorpos de Cadeia Única/genética , Biologia Sintética , Afinidade de Anticorpos , Receptor gp130 de Citocina/genética , Epitopos , Genes Reporter , Células HEK293 , Humanos , Ligantes , Mutação , Domínios e Motivos de Interação entre Proteínas , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptores Artificiais/metabolismo , Receptores da Eritropoetina/metabolismo , Transdução de Sinais , Anticorpos de Cadeia Única/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética
16.
Methods Mol Biol ; 2312: 89-107, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34228286

RESUMO

The quest to engineer increasingly complex synthetic gene networks in mammalian and plant cells requires an ever-growing portfolio of orthogonal gene expression systems. To control gene expression, light is of particular interest due to high spatial and temporal resolution, ease of dosage and simplicity of administration, enabling increasingly sophisticated man-machine interfaces. However, the majority of applied optogenetic switches are crowded in the UVB, blue and red/far-red light parts of the optical spectrum, limiting the number of simultaneously applicable stimuli. This problem is even more pertinent in plant cells, in which UV-A/B, blue, and red light-responsive photoreceptors are already expressed endogenously. To alleviate these challenges, we developed a green light responsive gene switch, based on the light-sensitive bacterial transcription factor CarH from Thermus thermophilus and its cognate DNA operator sequence CarO. The switch is characterized by high reversibility, high transgene expression levels, and low leakiness, leading to up to 350-fold induction ratios in mammalian cells. In this chapter, we describe the essential steps to build functional components of the green light-regulated gene switch, followed by detailed protocols to quantify transgene expression over time in mammalian cells. In addition, we expand this protocol with a description of how the optogenetic switch can be implemented in protoplasts of A. thaliana.


Assuntos
Arabidopsis/efeitos da radiação , Proteínas de Bactérias/efeitos da radiação , Engenharia Celular , Genes de Troca , Luz , Optogenética , Plantas Geneticamente Modificadas/efeitos da radiação , Thermus thermophilus/genética , Animais , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Técnicas de Cultura de Células , Células Cultivadas , Regulação Bacteriana da Expressão Gênica/efeitos da radiação , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Genes Reporter , Humanos , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Thermus thermophilus/metabolismo , Fatores de Tempo , Transfecção
17.
Methods Mol Biol ; 2312: 237-251, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34228294

RESUMO

Chemical control of protein localization is a powerful approach for manipulating mammalian cellular processes. Self-localizing ligand-induced protein translocation (SLIPT) is an emerging platform that enables control of protein localization in living mammalian cells using synthetic self-localizing ligands (SLs). We recently established a chemogenetic SLIPT system, in which any protein of interest fused to an engineered variant of Escherichia coli dihydrofolate reductase, DHFRiK6, can be rapidly and specifically translocated from the cytoplasm to the inner leaflet of the plasma membrane (PM) using a trimethoprim (TMP)-based PM-targeting SL, mDcTMP. The mDcTMP-mediated PM recruitment of DHFRiK6-fusion proteins can be efficiently returned to the cytoplasm by subsequent addition of free TMP, enabling temporal and reversible control over the protein localization. Here we describe the use of this mDcTMP/DHFRiK6-based SLIPT system for inducing (1) reversible protein translocation and (2) synthetic activation of the Raf/ERK pathway. This system provides a simple and versatile tool in mammalian synthetic biology for temporally manipulating various signaling molecules and pathways at the PM.


Assuntos
Engenharia Celular , Proteínas de Escherichia coli/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas Genéticas , Biologia Sintética , Tetra-Hidrofolato Desidrogenase/genética , Trimetoprima/farmacologia , Técnicas de Cultura de Células , Membrana Celular/metabolismo , Proteínas de Escherichia coli/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Microscopia de Fluorescência , Transporte Proteico , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Tetra-Hidrofolato Desidrogenase/metabolismo , Quinases raf/metabolismo
18.
Methods Mol Biol ; 2312: 321-328, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34228300

RESUMO

Single-cell engineering via virus based genetic manipulation allows the possibility of understanding of complex tissues. However, current delivery methods for the genetic engineering of single cells via viral transduction suffer from limitations that restrict their application. Here I present a protocol describing a precise technique which can be used for the targeted virus infection of single cells in a monolayer of cells that is optically accessible. The protocol, demonstrated here by stamping cultured Hela cells with lentiviruses (LVs), completes in a few minutes and allows stable transgene expression within a few days, at success rates approaching 80%.


Assuntos
Engenharia Celular , Vetores Genéticos , Lentivirus/genética , Magnetismo , Nanopartículas de Magnetita , Análise de Célula Única , Transdução Genética , Técnicas de Cultura de Células , Regulação Neoplásica da Expressão Gênica , Genes Reporter , Células HeLa , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência
19.
Methods Mol Biol ; 2312: 309-320, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34228299

RESUMO

Developments in genome-editing technology, especially CRISPR-Cas9, have revolutionized the way in which genetically engineered animals are generated. However, the process of generation includes microinjection to the one-cell stage embryo and the transfer of the microinjected embryo to the surrogate animals, which requires trained personnel. We recently reported the method includes introduction of CRISPR-Cas9 systems to the developing cerebral cortex via in utero electroporation thus generating gene-targeted neural stem cells in vivo. This technique is widely applicable for gene knockout, monitoring gene expression, and lineage analysis in developmental biology. In this chapter, the detailed protocol of EGFP (enhanced green fluorescent protein) knock-in method via in utero electroporation is described.


Assuntos
Encéfalo/metabolismo , Eletroporação , Regulação da Expressão Gênica no Desenvolvimento , Marcação de Genes , Técnicas de Transferência de Genes , Animais , Encéfalo/embriologia , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Feminino , Técnicas de Introdução de Genes , Genes Reporter , Idade Gestacional , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Gravidez , RNA Guia/genética , RNA Guia/metabolismo
20.
Methods Mol Biol ; 2319: 119-136, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34331250

RESUMO

We describe a novel, efficient method to identify cis-acting DNA sequences that drive cell-specific gene expression during development. We utilize transfer of Bacterial Artificial Chromosome (BAC) genomic DNAs, modified to contain a reporter gene, into fertilized mouse embryos and placing the injected embryos into pseudopregnant recipient females. The embryos are allowed to develop in utero for defined times after which they are collected for analysis. Using DNAs containing the LacZ reporter gene facilitates the analysis of gene activity through microscopy of intact embryos and subsequent sectioning of the stained embryos. With this technique cis-element activity can be identified and evaluated through further mutational analysis of the injected BAC DNA. This allows the identification of important gene regulatory domains that specify stage-specific gene expression in the developing embryo.


Assuntos
Cromossomos Artificiais Bacterianos/genética , Embrião de Mamíferos/diagnóstico por imagem , Sequências Reguladoras de Ácido Nucleico/genética , Animais , Embrião de Mamíferos/metabolismo , Feminino , Genes Reporter/genética , Camundongos , Camundongos Transgênicos , Microinjeções/métodos , Recombinação Genética , beta-Galactosidase/genética
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