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1.
J Toxicol Sci ; 47(9): 359-373, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36047110

RESUMO

Hepatotoxicity is one of the most common toxicities observed in non-clinical safety studies of drug candidates, and it is important to understand the hepatotoxicity mechanism to assess the risk of drug-induced liver injury in humans. In this study, we investigated the mechanism of hepatotoxicity caused by 2-[2-Methyl-1-(oxan-4-yl)-1H-benzimidazol-5-yl]-1,3-benzoxazole (DSP-0640), a drug candidate that showed hepatotoxicity characterized by centrilobular hypertrophy and vacuolation of hepatocytes in a 4-week oral repeated-dose toxicity study in male rats. In the liver of rats treated with DSP-0640, the expression of aryl hydrocarbon receptor (AHR) target genes, including Cyp1a1, was upregulated. In in vitro reporter assays, however, DSP-0640 showed only minimal AHR-activating potency. Therefore, we investigated the possibility that DSP-0640 indirectly activated AHR by inhibiting the CYP1 enzyme-dependent clearance of endogenous AHR agonists. In in vitro assays, DSP-0640 showed inhibitory effects on both rat and human CYP1A1 and enhanced rat and human AHR-mediated reporter gene expression induced by 6-formylindolo[3,2-b]carbazole, a well-known endogenous AHR agonist. The possible involvement of CYP1A1 inhibition in AHR activation was also demonstrated with other hepatotoxic compounds tacrine and albendazole. These results suggest that CYP1A1 inhibition-mediated AHR activation is involved in the hepatotoxicity caused by DSP-0640 and that DSP-0640 might induce hepatotoxicity in humans as well. We propose that CYP1A1 inhibition-mediated AHR activation is a novel mechanism for drug-induced hepatotoxicity.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas , Citocromo P-450 CYP1A1 , Receptores de Hidrocarboneto Arílico , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Doença Hepática Induzida por Substâncias e Drogas/genética , Citocromo P-450 CYP1A1/metabolismo , Genes Reporter , Hepatócitos/metabolismo , Humanos , Masculino , Ratos , Receptores de Hidrocarboneto Arílico/metabolismo
2.
Int J Mol Sci ; 23(16)2022 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-36012634

RESUMO

Previously, we established a highly sensitive promoter-trapping vector system using the piggyBac transposon for the efficient isolation of reporter cells. Herein, we examine whether this screening system can be applied to obtain vitamin-responsive cells. As a result, one and two reporter cells that responded to bexarotene (vitamin A) and calcitriol (vitamin D), respectively, were isolated from 4.7 × 106 seeded HeLaS3 cells. 5' RACE analyses identified the well-known CYP24A1 gene as a calcitriol-responsive gene, as well as two new bexarotene- or calcitriol-responsive genes, BDKRB2 and TSKU, respectively. TSKU, interestingly, also responded to bexarotene. Endogenous levels of the TSKU and BDKRB2 transcripts displayed only slight changes and were not detected in the comprehensive analyses performed to date. Dose-response analyses of BDKRB2 and TSKU reporter cells in parallel revealed a differential profile in response to each vitamin A agonist, suggesting a bioanalyzer. The present study demonstrates that producing multiple reporter cells by a type of random screening can efficiently identify novel genes with unusual characteristics and be used for the profiling of the properties of vitamin compounds. Similar approaches to the method shown here may be useful for identifying new markers and for the analysis or diagnosis of nutrients, toxins, metabolites, etc.


Assuntos
Calcitriol , Vitamina A , Bexaroteno , Calcitriol/metabolismo , Genes Reporter , Regiões Promotoras Genéticas , Receptores de Calcitriol/metabolismo , Vitamina A/farmacologia , Vitaminas/farmacologia
3.
Proc Natl Acad Sci U S A ; 119(35): e2122004119, 2022 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-35994666

RESUMO

Premature termination codons (PTCs) account for 10 to 20% of genetic diseases in humans. The gene inactivation resulting from PTCs can be counteracted by the use of drugs stimulating PTC readthrough, thereby restoring production of the full-length protein. However, a greater chemical variety of readthrough inducers is required to broaden the medical applications of this therapeutic strategy. In this study, we developed a reporter cell line and performed high-throughput screening (HTS) to identify potential readthrough inducers. After three successive assays, we isolated 2-guanidino-quinazoline (TLN468). We assessed the clinical potential of this drug as a potent readthrough inducer on the 40 PTCs most frequently responsible for Duchenne muscular dystrophy (DMD). We found that TLN468 was more efficient than gentamicin, and acted on a broader range of sequences, without inducing the readthrough of normal stop codons (TC).


Assuntos
Códon sem Sentido , Doenças Genéticas Inatas , Guanidinas , Quinazolinas , Linhagem Celular , Códon sem Sentido/efeitos dos fármacos , Códon sem Sentido/genética , Códon de Terminação/efeitos dos fármacos , Códon de Terminação/genética , Avaliação Pré-Clínica de Medicamentos , Genes Reporter/efeitos dos fármacos , Doenças Genéticas Inatas/tratamento farmacológico , Doenças Genéticas Inatas/genética , Gentamicinas/farmacologia , Guanidinas/farmacologia , Ensaios de Triagem em Larga Escala , Humanos , Distrofia Muscular de Duchenne/tratamento farmacológico , Distrofia Muscular de Duchenne/genética , Quinazolinas/farmacologia
4.
Int J Mol Sci ; 23(15)2022 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-35955578

RESUMO

The use of molecular imaging technologies for brain imaging can not only play an important supporting role in disease diagnosis and treatment but can also be used to deeply study brain functions. Recently, with the support of reporter gene technology, optical imaging has achieved a breakthrough in brain function studies at the molecular level. Reporter gene technology based on traditional clinical imaging modalities is also expanding. By benefiting from the deeper imaging depths and wider imaging ranges now possible, these methods have led to breakthroughs in preclinical and clinical research. This article focuses on the applications of magnetic resonance imaging (MRI), single-photon emission computed tomography (SPECT), and positron emission tomography (PET) reporter gene technologies for use in brain imaging. The tracking of cell therapies and gene therapies is the most successful and widely used application of these techniques. Meanwhile, breakthroughs have been achieved in the research and development of reporter genes and their imaging probe pairs with respect to brain function research. This paper introduces the imaging principles and classifications of the reporter gene technologies of these imaging modalities, lists the relevant brain imaging applications, reviews their characteristics, and discusses the opportunities and challenges faced by clinical imaging modalities based on reporter gene technology. The conclusion is provided in the last section.


Assuntos
Tomografia por Emissão de Pósitrons , Tomografia Computadorizada por Raios X , Encéfalo/diagnóstico por imagem , Genes Reporter , Imageamento por Ressonância Magnética , Neuroimagem , Tomografia por Emissão de Pósitrons/métodos , Tomografia Computadorizada de Emissão de Fóton Único/métodos
5.
Science ; 377(6608): eabi8654, 2022 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-35981026

RESUMO

Predicting the function of noncoding variation is a major challenge in modern genetics. In this study, we used massively parallel reporter assays to screen 5706 variants identified from genome-wide association studies for both Alzheimer's disease (AD) and progressive supranuclear palsy (PSP), identifying 320 functional regulatory variants (frVars) across 27 loci, including the complex 17q21.31 region. We identified and validated multiple risk loci using CRISPR interference or excision, including complement 4 (C4A) and APOC1 in AD and PLEKHM1 and KANSL1 in PSP. Functional variants disrupt transcription factor binding sites converging on enhancers with cell type-specific activity in PSP and AD, implicating a neuronal SP1-driven regulatory network in PSP pathogenesis. These analyses suggest that noncoding genetic risk is driven by common genetic variants through their aggregate activity on specific transcriptional programs.


Assuntos
Doença de Alzheimer , Cromossomos Humanos Par 17 , Redes Reguladoras de Genes , Variação Genética , Regiões não Traduzidas , Doença de Alzheimer/genética , Cromossomos Humanos Par 17/genética , Genes Reporter , Loci Gênicos , Estudo de Associação Genômica Ampla , Humanos , Fatores de Risco , Paralisia Supranuclear Progressiva/genética , Regiões não Traduzidas/genética
6.
Methods Mol Biol ; 2524: 223-233, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35821475

RESUMO

Reverse genetics systems provide a powerful tool to generate recombinant arenavirus expressing reporters to facilitate the investigation of the arenavirus life cycle and also for the discovery of antiviral countermeasures. The plasmid-encoded viral ribonucleoprotein components initiate the transcription and replication of a plasmid-driven full-length viral genome, resulting in infectious virus. Thereby, this approach is ideal for the generation of recombinant arenaviruses expressing reporter genes that can be used as valid surrogates for virus replication. By splitting the small viral segment (S) into two viral segments (S1 and S2), each of them encoding a reporter gene, recombinant tri-segmented arenavirus can be rescued. Bi-reporter-expressing recombinant tri-segmented arenaviruses represent an excellent tool to study the biology of arenaviruses, including the identification and characterization of both prophylactic and therapeutic countermeasures for the treatment of arenaviral infections. In this chapter, we describe a detailed protocol on the generation and in vitro characterization of recombinant arenaviruses containing a tri-segment genome expressing two reporter genes based on the prototype member in the family, lymphocytic choriomeningitis virus (LCMV). Similar experimental approaches can be used for the generation of bi-reporter-expressing tri-segment recombinant viruses for other members in the arenavirus family.


Assuntos
Infecções por Arenaviridae , Genética Reversa , Infecções por Arenaviridae/genética , Infecções por Arenaviridae/prevenção & controle , Genes Reporter , Humanos , Vírus da Coriomeningite Linfocítica/genética , Genética Reversa/métodos , Replicação Viral/genética
7.
Methods Mol Biol ; 2524: 409-432, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35821490

RESUMO

Multiplex hextuple luciferase assaying allows monitoring the activity of five experimental pathways against one control at the same time. To perform multiplex hextuple luciferase assaying, six orthogonal luciferase reporter units are needed of which five are pathway-specific and one acts as a control for normalization. To ensure stoichiometric delivery of all six luciferase reporters in every transfected cell, synthetic assembly DNA cloning is used to stitch together all six luciferase reporter units into a single vector. Here, we provide a detailed three-step synthetic assembly DNA protocol to generate multiplex hextuple luciferase reporter plasmids for any five cellular signaling pathways of interest, against a control normalization pathway. A first protocol is provided on how to generate plasmids that contain novel transcription factor-binding motifs for specific transcription factors. A second protocol details on how to couple these novel transcription factor-binding motifs to one of five orthogonal luciferases to obtain specific luciferase reporters for cellular signaling pathways acting upstream of those transcription factor-binding motifs. Finally, a third protocol provides details on how to assemble orthogonal luciferase reporters for five cellular signaling pathways acting upstream of five unique transcription factor-binding motifs together with a control constitutive pathway luciferase reporter that will be used for normalization to obtain a final multiplex hextuple luciferase vector.


Assuntos
DNA , Fatores de Transcrição , Clonagem Molecular , DNA/genética , Genes Reporter , Luciferases/genética , Plasmídeos/genética , Fatores de Transcrição/metabolismo
8.
Proc Natl Acad Sci U S A ; 119(27): e2202862119, 2022 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-35776547

RESUMO

Identifying the genetic basis of repeatedly evolved traits provides a way to reconstruct their evolutionary history and ultimately investigate the predictability of evolution. Here, we focus on the oldfield mouse (Peromyscus polionotus), which occurs in the southeastern United States, where it exhibits considerable color variation. Dorsal coats range from dark brown in mainland mice to near white in mice inhabiting sandy beaches; this light pelage has evolved independently on Florida's Gulf and Atlantic coasts as camouflage from predators. To facilitate genomic analyses, we first generated a chromosome-level genome assembly of Peromyscus polionotus subgriseus. Next, in a uniquely variable mainland population (Peromyscus polionotus albifrons), we scored 23 pigment traits and performed targeted resequencing in 168 mice. We find that pigment variation is strongly associated with an ∼2-kb region ∼5 kb upstream of the Agouti signaling protein coding region. Using a reporter-gene assay, we demonstrate that this regulatory region contains an enhancer that drives expression in the dermis of mouse embryos during the establishment of pigment prepatterns. Moreover, extended tracts of homozygosity in this Agouti region indicate that the light allele experienced recent and strong positive selection. Notably, this same light allele appears fixed in both Gulf and Atlantic coast beach mice, despite these populations being separated by >1,000 km. Together, our results suggest that this identified Agouti enhancer allele has been maintained in mainland populations as standing genetic variation and from there, has spread to and been selected in two independent beach mouse lineages, thereby facilitating their rapid and parallel evolution.


Assuntos
Proteína Agouti Sinalizadora , Evolução Biológica , Elementos Facilitadores Genéticos , Peromyscus , Pigmentação da Pele , Proteína Agouti Sinalizadora/metabolismo , Alelos , Animais , Genes Reporter , Peromyscus/genética , Peromyscus/fisiologia , Pigmentação da Pele/genética
9.
BMC Plant Biol ; 22(1): 356, 2022 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-35864445

RESUMO

BACKGROUND: For the needs of modern biotechnology, a quantitative approach to the control of regulatory elements at all stages of gene expression has long become indispensable. Such a control regime is impossible without a quantitative analysis of the role of each regulatory element or pattern used. Therefore, it seems important to modify and develop the accuracy, reproducibility, and availability of methods for quantifying the contribution of each regulatory code to the implementation of genetic information. RESULTS: A new vector system for transient expression in plants is described; this system is intended for quantitative analysis of the contribution of regulatory elements to transcription and translation efficiencies. The proposed vector comprises two expression cassettes carrying reporter genes (of the Clostridium thermocellum thermostable lichenase and E. coli ß-glucuronidase) under the control of different promoters. Herewith we also propose a new method for quantification of the effect of tested regulatory elements on expression, which relies on assessment of the enzyme activities of reporter proteins taking into account the transcription of their genes. CONCLUSIONS: In our view, this approach makes it possible to precisely determine the amounts of reporter proteins and their transcripts at all stages of expression. The efficiency of the proposed system has been validated by the analysis of the roles of known translation enhancers at the stages of transcription and translation.


Assuntos
Escherichia coli , Sequências Reguladoras de Ácido Nucleico , Escherichia coli/genética , Genes Reporter , Regiões Promotoras Genéticas/genética , Sequências Reguladoras de Ácido Nucleico/genética , Reprodutibilidade dos Testes
10.
Methods Mol Biol ; 2537: 211-229, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35895267

RESUMO

Alternative splicing is a key layer of gene regulation that is frequently modulated in a spatiotemporal manner. As such, it is a major goal to understand the mechanisms controlling alternative splicing in specific cellular contexts. Reporters that recapitulate alternative splicing patterns of endogenous transcripts have served as excellent tools for dissecting regulatory mechanisms of splicing. In this chapter, we describe a two-color fluorescent reporter system that enables the visualization of alternative splicing patterns by microscopy at single-cell resolution in live animals. We present this reporter system in the context of the model nematode C. elegans.


Assuntos
Processamento Alternativo , Caenorhabditis elegans , Animais , Caenorhabditis elegans/genética , Genes Reporter , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética
11.
Viruses ; 14(7)2022 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-35891336

RESUMO

Membrane fusion constitutes an essential step in the replication cycle of numerous viral pathogens, hence it represents an important druggable target. In the present study, we established a virus-free, stable reporter fusion inhibition assay (SRFIA) specifically designed to identify compounds interfering with virus-induced membrane fusion. The dual reporter assay is based on two stable Vero cell lines harboring the third-generation tetracycline (Tet3G) transactivator and a bicistronic reporter gene cassette under the control of the tetracycline responsive element (TRE3G), respectively. Cell-cell fusion by the transient transfection of viral fusogens in the presence of doxycycline results in the expression of the reporter enzyme secreted alkaline phosphatase (SEAP) and the fluorescent nuclear localization marker EYFPNuc. A constitutively expressed, secreted form of nanoluciferase (secNLuc) functioned as the internal control. The performance of the SRFIA was tested for the quantification of SARS-CoV-2- and HSV-1-induced cell-cell fusion, respectively, showing high sensitivity and specificity, as well as the reliable identification of known fusion inhibitors. Parallel quantification of secNLuc enabled the detection of cytotoxic compounds or insufficient transfection efficacy. In conclusion, the SRFIA reported here is well suited for high-throughput screening for new antiviral agents and essentially will be applicable to all viral fusogens causing cell-cell fusion in Vero cells.


Assuntos
COVID-19 , Herpesvirus Humano 1 , Animais , Antivirais/farmacologia , Linhagem Celular , Chlorocebus aethiops , Genes Reporter , Herpesvirus Humano 1/genética , Humanos , Fusão de Membrana , SARS-CoV-2/genética , Tetraciclinas , Células Vero
12.
Sci Rep ; 12(1): 11489, 2022 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-35798760

RESUMO

Luciferases are often used as a sensitive, versatile reporter in cell-free transcription-translation (TXTL) systems, for research and practical applications such as engineering genetic parts, validating genetic circuits, and biosensor outputs. Currently, only two luciferases (Firefly and Renilla) are commonly used without substrate cross-talk. Here we demonstrate the expansion of the cell-free luciferase reporter system, with two orthogonal luciferase reporters: N. nambi luciferase (Luz) and LuxAB. These luciferases do not have cross-reactivity with the Firefly and Renilla substrates. We also demonstrate a substrate regeneration pathway for one of the new luciferases, enabling long-term time courses of protein expression monitoring in the cell-free system. Furthermore, we reduced the number of genes required in TXTL expression, by engineering a cell extract containing part of the luciferase enzymes. Our findings lead to an expanded platform with multiple orthogonal luminescence translation readouts for in vitro protein expression.


Assuntos
Vaga-Lumes , Luminescência , Animais , Vaga-Lumes/genética , Vaga-Lumes/metabolismo , Genes Reporter , Indicadores e Reagentes , Luciferases/genética , Luciferases/metabolismo , Luciferases de Vaga-Lume/genética
13.
J Toxicol Sci ; 47(6): 249-255, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35650141

RESUMO

Retinoic acid, an active form of vitamin A, plays very important roles in mammalian embryogenesis. The concentration of retinoic acid is extremely low and strictly regulated by enzymes of cytochrome P450 (CYP) family, CYP26s (CYP26A1, CYP26B1 and CYP26C1) in the cells. Therefore, it is thought that changes in CYP26s activities due to exposure to a wide variety of drugs and chemicals exhibit teratogenicity. In this study, to easily detect the changes in retinoic acid level, we constructed an adenovirus-mediated reporter assay system using the promoter region of the CYP26A1 gene and inserting retinoic acid response element (RARE) and retinoid X response element (RXRE) into the downstream of the luciferase gene of reporter plasmid, which highly increased the response to retinoic acid. Reporter activity significantly increased in a concentration-dependent manner with retinoic acid; this increase was also observed at least after treatment with a very low concentration of 1 nM retinoic acid. This increase was suppressed by the accelerated metabolism of retinoic acid due to the overexpression of CYP26A1; however, this suppression was almost completely suspended by treatment with talarozole, a CYP26 inhibitor. In conclusion, the reporter assay system constructed using the induction of CYP26A1 expression is a risk assessment system that responds to extremely low concentrations of retinoic acid and is useful for assessing the excess vitamin A mediated teratogenicity caused by various chemicals at the cellular level.


Assuntos
Adenoviridae , Teratógenos , Tretinoína , Adenoviridae/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Genes Reporter , Humanos , Células MCF-7 , Regiões Promotoras Genéticas , Ácido Retinoico 4 Hidroxilase/genética , Teratógenos/análise , Tretinoína/análise , Vitamina A
14.
Arch Toxicol ; 96(9): 2501-2510, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35678845

RESUMO

The activity of nuclear receptors (e.g., pregnane x receptor, PXR) can be assessed by luminescence-based dual reporter gene assays. Under most conditions, receptor-activated firefly luminescence is normalized to Renilla luminescence, which is triggered by a constitutively active promoter. Simultaneous damage to the cells can however disrupt these signals and thus impair the interpretation of the data. Consequently, this study addressed three important aspects: First, idealized models were described, each highlighting crucial characteristics and important pitfalls of dual PXR reporter gene assays used to evaluate PXR activation or inhibition. Second, these models were supported by experimental data obtained with a strong PXR activator (rifampicin) with low cytotoxicity, a PXR activator with high cytotoxicity (dovitinib), a proposed PXR inhibitor that reportedly has no toxic effects (triptolide), and a cytotoxic control (oxaliplatin). Data were evaluated for relative PXR activity data, individual firefly or Renilla luminescence, and anti-proliferative effects of the compounds (assessed by crystal violet staining). Finally, a step-by-step guide is proposed to avoid misleading set-up of the assay or misinterpretation of the data obtained. Key considerations here include (1) omission of drug concentrations beyond 10-20% proliferation inhibition; (2) observation of Renilla luminescence, because this tends to indicate 'false PXR activation' when it inexplicably decreases; (3) parallel decrease of relative PXR activity and proliferation below baseline levels in conjunction with a sharp decrease in Renilla luminescence indicates 'false PXR antagonism'; (4) non-sigmoidal relationships suggest the absence of concentration dependency.


Assuntos
Receptores de Esteroides , Citocromo P-450 CYP3A/genética , Genes Reporter , Receptores Citoplasmáticos e Nucleares/genética , Receptores de Esteroides/genética , Rifampina/farmacologia
15.
Sci Rep ; 12(1): 9844, 2022 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-35701460

RESUMO

Chikungunya virus (CHIKV), a highly infectious and rapidly spread viral pathogen, is classified as a pathogenic agent at the biosafety level 3. Operation of live authentic CHIKV needs a specific laboratory with the P3 or above containment, which greatly confines the CHIKV-associated studies. To establish an evaluation system of CHIKV that can be utilized in a BSL2 laboratory, we constructed a pseudovirus (PsV) system of CHIKV containing double reporter genes (ZsGreen1 and luciferase). The fluorescent ZsGreen1 is a convenient and cheap reporter for monitoring the efficiency of transfection and titration of PsV. The enzyme luciferase is a sensitive reporter for the application of PsV to neutralization assay or drug screening. The CHIKV PsV produced in this study, with a titer of up to 3.16 × 106 TU/ml, was confirmed by Western blotting and transmission electronic microscopy (TEM). Finally, we developed a microneutralization assay with the CHIKV PsV produced in this study, which was successfully applied to evaluate neutralizing activities of convalescent sera from CHIKV-infected patients. In summary, we have established a convenient and sensitive double-reporter CHIKV pseudovirus system, which provides a safe and effective platform for screening anti-CHIKV drugs and evaluating vaccines against CHIKV.


Assuntos
Febre de Chikungunya , Vírus Chikungunya , Anticorpos Neutralizantes , Anticorpos Antivirais , Febre de Chikungunya/diagnóstico , Vírus Chikungunya/genética , Genes Reporter , Humanos , Luciferases/genética , Testes de Neutralização
16.
Sci Rep ; 12(1): 9874, 2022 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-35701501

RESUMO

Stem cell therapy has great potential for replacing beta-cell loss in diabetic patients. However, a key obstacle to cell therapy's success is to preserve viability and function of the engrafted cells. While several strategies have been developed to improve engrafted beta-cell survival, tools to evaluate the efficacy within the body by imaging are limited. Traditional labeling tools, such as GFP-like fluorescent proteins, have limited penetration depths in vivo due to tissue scattering and absorption. To circumvent this limitation, a near-infrared fluorescent mutant version of the DrBphP bacteriophytochrome, iRFP720, has been developed for in vivo imaging and stem/progenitor cell tracking. Here, we present the generation and characterization of an iRFP720 expressing human induced pluripotent stem cell (iPSC) line, which can be used for real-time imaging in various biological applications. To generate the transgenic cells, the CRISPR/Cas9 technology was applied. A puromycin resistance gene was inserted into the AAVS1 locus, driven by the endogenous PPP1R12C promoter, along with the CAG-iRFP720 reporter cassette, which was flanked by insulator elements. Proper integration of the transgene into the targeted genomic region was assessed by comprehensive genetic analysis, verifying precise genome editing. Stable expression of iRFP720 in the cells was confirmed and imaged by their near-infrared fluorescence. We demonstrated that the reporter iPSCs exhibit normal stem cell characteristics and can be efficiently differentiated towards the pancreatic lineage. As the genetically modified reporter cells show retained pluripotency and multilineage differentiation potential, they hold great potential as a cellular model in a variety of biological and pharmacological applications.


Assuntos
Células-Tronco Pluripotentes Induzidas , Diferenciação Celular/genética , Edição de Genes , Genes Reporter , Humanos , Regiões Promotoras Genéticas , Transgenes
17.
Biologicals ; 77: 16-23, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35729037

RESUMO

To develop and validate a novel reporter gene assay (RGA) to detect pyrogen, HL60 cells were transfected with an NF-κB-RE plasmid containing the luciferase gene to generate stably transfected cells. Through stimulation with pyrogens, a signal was obtained that was dose-dependent with the concentration of pyrogen. Using the cells, we selected and optimized the parameters and found that the optimal conditions may be with 5 × 105/ml cells that were seeded and incubated with pyrogen for 3-6 h in IMDM medium with 2% FBS. Based on the optimized parameters, a novel RGA was developed. Then, the RGA was validated and the results showed that the linearity was greater than 0.95 between the signals and the concentrations of pyrogen, the recoveries of pyrogen were all between 50% and 200%, and the precision was less than 35%. There was no difference in the sensitivity, specificity or reproducibility between RGA and BET, and the results from RGA and MAT and RPT were consistent. Furthermore, the RGA can be applied to the pyrogen detection of monoclonal antibodies. Due to its advantages including a fast detection speed, high sensitivity, convenient mode of operation and wide-pyrogen spectrum detection, RGA is promising as a supplementary method to detect pyrogen.


Assuntos
Bioensaio , Pirogênios , Bioensaio/métodos , Genes Reporter , Luciferases/genética , Reprodutibilidade dos Testes
18.
Microb Cell Fact ; 21(1): 107, 2022 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-35655187

RESUMO

BACKGROUND: Cordyceps militaris, a kind of edible and medicinal fungus widely accepted in East Asia, has attracted much attention as a potential cell factory for producing adenosine analogs. Despite the rapid development in gene editing techniques and genome modeling, the diversity of DNA elements in C. militaris was too short to achieve rational heterogeneous expression for metabolic engineering studies. RESULTS: In this study, PtrpC, a kind of promoter with a relatively appropriate expression level and small size, was selected as a monomer for promoter library construction. Through in vitro BioBricks assembly, 9 overlapping PtrpC promoters with different copy numbers as well as reporter gene gfp were connected and subsequently integrated into the genome of C. militaris. Both the mRNA transcription level and the expression level of gene gfp gradually increased along with the copy number of the overlapping promoter NPtrpC and peaked at 7. In the meantime, no significant difference was found in either the biomass or morphological characteristic of engineered and wild-type strains. CONCLUSIONS: This study firstly expanded the overlapping promoter strategy used in model microorganism in C. militaris. It was a proof-of-concept in fungi synthetic biology and provide a general method to pushed the boundary of promoter engineering in edible mushroom.


Assuntos
Cordyceps , Clonagem Molecular , Cordyceps/genética , Biblioteca Gênica , Genes Reporter , Regiões Promotoras Genéticas
19.
Development ; 149(14)2022 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-35713287

RESUMO

Biological systems are increasingly viewed through a quantitative lens that demands accurate measures of gene expression and local protein concentrations. CRISPR/Cas9 gene tagging has enabled increased use of fluorescence to monitor proteins at or near endogenous levels under native regulatory control. However, owing to typically lower expression levels, experiments using endogenously tagged genes run into limits imposed by autofluorescence (AF). AF is often a particular challenge in wavelengths occupied by commonly used fluorescent proteins (GFP, mNeonGreen). Stimulated by our work in C. elegans, we describe and validate Spectral Autofluorescence Image Correction By Regression (SAIBR), a simple platform-independent protocol and FIJI plug-in to correct for autofluorescence using standard filter sets and illumination conditions. Validated for use in C. elegans embryos, starfish oocytes and fission yeast, SAIBR is ideal for samples with a single dominant AF source; it achieves accurate quantitation of fluorophore signal, and enables reliable detection and quantification of even weakly expressed proteins. Thus, SAIBR provides a highly accessible low-barrier way to incorporate AF correction as standard for researchers working on a broad variety of cell and developmental systems.


Assuntos
Caenorhabditis elegans , Proteínas , Animais , Fluorescência , Corantes Fluorescentes , Genes Reporter
20.
Int J Mol Sci ; 23(11)2022 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-35683022

RESUMO

Optogenetics has the advantages of a fast response time, reversibility, and high spatial and temporal resolution, which make it desirable in the metabolic engineering of chassis cells. In this study, a light-induced expression system of Yarrowia lipolytica was constructed, which successfully achieved the synthesis and functional verification of Bleomycin resistance protein (BleoR). The core of the blue light-induced system, the light-responsive element (TF), is constructed based on the blue photosensitive protein EL222 and the transcription activator VP16. The results show that the light-induced sensor based on TF, upstream activation sequence (C120)5, and minimal promoter CYC102 can respond to blue light and initiate the expression of GFPMut3 report gene. With four copies of the responsive promoter and reporter gene assembled, they can produce a 128.5-fold higher fluorescent signal than that under dark conditions after 8 h of induction. The effects of light dose and periodicity on this system were investigated, which proved that the system has good spatial and temporal controllability. On this basis, the light-controlled system was used for the synthesis of BleoR to realize the expression and verification of functional protein. These results demonstrated that this system has the potential for the transcriptional regulation of target genes, construction of large-scale synthetic networks, and overproduction of the desired product.


Assuntos
Yarrowia , Genes Reporter , Engenharia Metabólica/métodos , Optogenética/métodos , Regiões Promotoras Genéticas , Yarrowia/genética , Yarrowia/metabolismo
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