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Nature Genetics Feb 2023Massively parallel reporter gene assays are key tools in regulatory genomics but cannot be used to identify cell-type-specific regulatory elements without performing...
Massively parallel reporter gene assays are key tools in regulatory genomics but cannot be used to identify cell-type-specific regulatory elements without performing assays serially across different cell types. To address this problem, we developed a single-cell massively parallel reporter assay (scMPRA) to measure the activity of libraries of cis-regulatory sequences (CRSs) across multiple cell types simultaneously. We assayed a library of core promoters in a mixture of HEK293 and K562 cells and showed that scMPRA is a reproducible, highly parallel, single-cell reporter gene assay that detects cell-type-specific cis-regulatory activity. We then measured a library of promoter variants across multiple cell types in live mouse retinas and showed that subtle genetic variants can produce cell-type-specific effects on cis-regulatory activity. We anticipate that scMPRA will be widely applicable for studying the role of CRSs across diverse cell types.
Topics: Animals; Humans; Mice; Gene Library; Genes, Reporter; HEK293 Cells; Promoter Regions, Genetic; Retina
PubMed: 36635387
DOI: 10.1038/s41588-022-01278-7 -
The Plant Cell Nov 1997A highly active synthetic auxin response element (AuxRE), referred to as DR5, was created by performing site-directed mutations in a natural composite AuxRE found in the...
A highly active synthetic auxin response element (AuxRE), referred to as DR5, was created by performing site-directed mutations in a natural composite AuxRE found in the soybean GH3 promoter. DR5 consisted of tandem direct repeats of 11 bp that included the auxin-responsive TGTCTC element. The DR5 AuxRE showed greater auxin responsiveness than a natural composite AuxRE and the GH3 promoter when assayed by transient expression in carrot protoplasts or in stably transformed Arabidopsis seedlings, and it provides a useful reporter gene for studying auxin-responsive transcription in wild-type plants and mutants. An auxin response transcription factor, ARF1, bound with specificity to the DR5 AuxRE in vitro and interacted with Aux/IAA proteins in a yeast two-hybrid system. Cotransfection experiments with natural and synthetic AuxRE reporter genes and effector genes encoding Aux/IAA proteins showed that overexpression of Aux/IAA proteins in carrot protoplasts resulted in specific repression of TGTCTC AuxRE reporter gene expression.
Topics: Arabidopsis; Gene Expression Regulation, Plant; Genes, Reporter; Glucuronidase; Indoleacetic Acids; Mutagenesis, Site-Directed; Plant Proteins; Plasmids; Promoter Regions, Genetic; Transfection
PubMed: 9401121
DOI: 10.1105/tpc.9.11.1963 -
International Journal of Molecular... Jul 2022The 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... (Review)
Review
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.
Topics: Brain; Genes, Reporter; Magnetic Resonance Imaging; Neuroimaging; Positron-Emission Tomography; Tomography, Emission-Computed, Single-Photon; Tomography, X-Ray Computed
PubMed: 35955578
DOI: 10.3390/ijms23158443 -
Theranostics 2018Molecular imaging has played an important role in the noninvasive exploration of multiple biological processes. Reporter gene imaging is a key part of molecular imaging.... (Review)
Review
Molecular imaging has played an important role in the noninvasive exploration of multiple biological processes. Reporter gene imaging is a key part of molecular imaging. By combining with a reporter probe, a reporter protein can induce the accumulation of specific signals that are detectable by an imaging device to provide indirect information of reporter gene expression in living subjects. There are many types of reporter genes and each corresponding imaging technique has its own advantages and drawbacks. Fused reporter genes or single reporter genes with products detectable by multiple imaging modalities can compensate for the disadvantages and potentiate the advantages of each modality. Reporter gene multimodality imaging could be applied to trace implanted cells, monitor gene therapy, assess endogenous molecular events, screen drugs, etc. Although several types of multimodality imaging apparatus and multimodality reporter genes are available, more sophisticated detectors and multimodality reporter gene systems are needed.
Topics: Animals; Cell Tracking; Drug Evaluation, Preclinical; Genes, Reporter; Genetic Therapy; Mice; Molecular Imaging; Multimodal Imaging; Neoplasms
PubMed: 29896296
DOI: 10.7150/thno.24108 -
Korean Journal of Radiology 2003Molecular imaging aims to visualize the cellular and molecular processes occurring in living tissues, and for the imaging of specific molecules in vivo, the development... (Review)
Review
Molecular imaging aims to visualize the cellular and molecular processes occurring in living tissues, and for the imaging of specific molecules in vivo, the development of reporter probes and dedicated imaging equipment is most important. Reporter genes can be used to monitor the delivery and magnitude of therapeutic gene transfer, and the time variation involved. Imaging technologies such as micro-PET, SPECT, MRI and CT, as well as optical imaging systems, are able to non-invasively detect, measure, and report the simultaneous expression of multiple meaningful genes. It is believed that recent advances in reporter probes, imaging technologies and gene transfer strategies will enhance the effectiveness of gene therapy trials.
Topics: Animals; Apoptosis; Contrast Media; Diagnostic Imaging; Gene Expression; Genes, Reporter; Genetic Therapy; Humans; Molecular Biology; Neovascularization, Physiologic; Rats
PubMed: 14726636
DOI: 10.3348/kjr.2003.4.4.201 -
BMC Molecular Biology Mar 2009The majority of the promoters, their regulatory elements, and their variations in the human genome remain unknown. Reporter gene technology for transcriptional activity...
BACKGROUND
The majority of the promoters, their regulatory elements, and their variations in the human genome remain unknown. Reporter gene technology for transcriptional activity is a widely used tool for the study of promoter structure, gene regulation, and signaling pathways. Construction of transcriptional reporter vectors, including use of cis-acting sequences, requires cloning and time-demanding manipulations, particularly with introduced mutations.
RESULTS
In this report, we describe a cloning-free strategy to generate transcriptionally-controllable linear reporter constructs. This approach was applied in common transcriptional models of inflammatory response and the interferon system. In addition, it was used to delineate minimal transcriptional activity of selected ribosomal protein promoters. The approach was tested for conversion of genes into TetO-inducible/repressible expression cassettes.
CONCLUSION
The simple introduction and tuning of any transcriptional control in the linear DNA product renders promoter activation and regulated gene studies simple and versatile.
Topics: Bacterial Proteins; Carrier Proteins; Cell Line; Cloning, Molecular; Gene Expression Regulation; Genes, Reporter; Humans; Models, Genetic
PubMed: 19267938
DOI: 10.1186/1471-2199-10-20 -
Journal of Nuclear Medicine : Official... Dec 2019The first reporter systems were developed in the early 1980s and were based on measuring the activity of an enzyme-as a surrogate measure of promoter-driven... (Review)
Review
The first reporter systems were developed in the early 1980s and were based on measuring the activity of an enzyme-as a surrogate measure of promoter-driven transcriptional activity-which is now known as a reporter gene system. The initial objective and application of reporter techniques was to analyze the activity of a specific promoter (namely, the expression of a gene that is under the regulation of the specific promoter that is linked to the reporter gene). This system allows visualization of specific promoter activity with great sensitivity. In general, there are 2 classes of reporter systems: constitutively expressed (always-on) reporter constructs used for cell tracking, and inducible reporter systems sensitive to endogenous signaling molecules and transcription factors that characterize specific tissues, tumors, or signaling pathways.This review traces the development of different reporter systems, using and proteins as well as reporter systems. The development and application of reporter systems is the focus of this review. The question at the end of the review is whether the "promise" of reporter gene imaging has been realized. What is required for moving forward with reporter systems, and what is required for successful translation to clinical applications?
Topics: Animals; Genes, Reporter; Humans; Molecular Imaging; Radionuclide Imaging
PubMed: 31792128
DOI: 10.2967/jnumed.118.220004 -
Molecules (Basel, Switzerland) May 2016Magnetic resonance imaging (MRI) is one of the most important imaging technologies used in clinical diagnosis. Reporter genes for MRI can be applied to accurately track... (Review)
Review
Magnetic resonance imaging (MRI) is one of the most important imaging technologies used in clinical diagnosis. Reporter genes for MRI can be applied to accurately track the delivery of cell in cell therapy, evaluate the therapy effect of gene delivery, and monitor tissue/cell-specific microenvironments. Commonly used reporter genes for MRI usually include genes encoding the enzyme (e.g., tyrosinase and β-galactosidase), the receptor on the cells (e.g., transferrin receptor), and endogenous reporter genes (e.g., ferritin reporter gene). However, low sensitivity limits the application of MRI and reporter gene-based multimodal imaging strategies are common including optical imaging and radionuclide imaging. These can significantly improve diagnostic efficiency and accelerate the development of new therapies.
Topics: Ferritins; Genes, Reporter; Genetic Therapy; Humans; Magnetic Resonance Imaging; Molecular Imaging; Multimodal Imaging
PubMed: 27213309
DOI: 10.3390/molecules21050580 -
Hellenic Journal of Nuclear Medicine 2017Reporter gene/probe systems have proved to be reliable for monitoring gene/cell therapy. We sought to evaluate whether a reporter gene/probe system, namely the human...
OBJECTIVE
Reporter gene/probe systems have proved to be reliable for monitoring gene/cell therapy. We sought to evaluate whether a reporter gene/probe system, namely the human estrogen receptor ligand binding domain (hERL)/16α-F fluoro-17β-estradiol (F-FES), could be used for monitoring vascular endothelial growth factor (VEGF) gene expression and response to bone marrow mesenchymal stem cell (MSCs) therapy in ischemic heart disease.
ANIMALS AND METHODS
Reporter gene hERL and therapeutic gene VEGF165 were linked through internal ribosome entry site (IRES), and then the recombinant adenovirus vector Adenovirus 5-hERL-IRES-VEGF (Ad5-EIV) was constructed and transfected into MSCs, and named Ad5-EIV-MSCs. Rat myocardial infarction was induced by coronary arterial branch ligature, and Ad5-EIV-MSCs were transplanted by injection into the peripheral myocardium, while non-transfected MSCs transplantation used as controls. Fluorine-18-FDG micro-PET imaging was performed to confirm myocardial infarction 1 day after surgery. Fluorine-18-FES micro-PET/CT images were acquired 2 days after Ad5-EIV-MSCs transplantation. Myocardial specimens were obtained and stained with hematoxylin-eosin (H&E) staining to verify the myocardial infarction. The expression of estrogen receptor (ER) and VEGF was detected using immunohistochemistry (IHC).
RESULTS
Rat myocardial infarction models were successfully produced and confirmed by H&E staining. Images of F-FDG PET showed obvious reduced or absent uptake of F-FDG on the infarct myocardium, while uniform and well-distribution on the normal myocardium. F-FES micro-PET/CT showed the tracer notable accumulated in the apical region where Ad5-EIV-MSCs were injected with an uptake value of 0.38±0.09%ID/g, which was much higher than that of surrounding normal myocardium with nearly no uptake of F-FES (0.10±0.03%ID/g, n=5, P<0.05). In the group of non-transfected MSCs, the apical uptake was similar to that of normal myocardium. Immunohistochemistry studies demonstrated positive expression of both ER and VEGF in the involved region accompanied by active angiogenesis.
CONCLUSION
This study confirmed that hERL/F-FES could be used as a reporter gene/probe system for monitoring gene and cell therapy in the ischemic heart disease.
Topics: Animals; Combined Modality Therapy; Fluorodeoxyglucose F18; Genes, Reporter; Genetic Therapy; Male; Mesenchymal Stem Cell Transplantation; Myocardial Ischemia; Positron Emission Tomography Computed Tomography; Radiopharmaceuticals; Rats; Rats, Sprague-Dawley; Treatment Outcome
PubMed: 29177255
DOI: 10.1967/s002449910601 -
World Journal of Gastroenterology Jul 2016Hepatocellular carcinoma (HCC) is one of the most common cancers in Eastern Asia, and its incidence is increasing globally. Numerous experimental models have been... (Review)
Review
Hepatocellular carcinoma (HCC) is one of the most common cancers in Eastern Asia, and its incidence is increasing globally. Numerous experimental models have been developed to better our understanding of the pathogenic mechanism of HCC and to evaluate novel therapeutic approaches. Molecular imaging is a convenient and up-to-date biomedical tool that enables the visualization, characterization and quantification of biologic processes in a living subject. Molecular imaging based on reporter gene expression, in particular, can elucidate tumor-specific events or processes by acquiring images of a reporter gene's expression driven by tumor-specific enhancers/promoters. In this review, we discuss the advantages and disadvantages of various experimental HCC mouse models and we present in vivo images of tumor-specific reporter gene expression driven by an alpha-fetoprotein (AFP) enhancer/promoter system in a mouse model of HCC. The current mouse models of HCC development are established by xenograft, carcinogen induction and genetic engineering, representing the spectrum of tumor-inducing factors and tumor locations. The imaging analysis approach of reporter genes driven by AFP enhancer/promoter is presented for these different HCC mouse models. Such molecular imaging can provide longitudinal information about carcinogenesis and tumor progression. We expect that clinical application of AFP-targeted reporter gene expression imaging systems will be useful for the detection of AFP-expressing HCC tumors and screening of increased/decreased AFP levels due to disease or drug treatment.
Topics: Animals; Carcinogens; Carcinoma, Hepatocellular; Disease Models, Animal; Enhancer Elements, Genetic; Gene Expression; Genes, Reporter; Genetic Engineering; Humans; Liver Neoplasms; Mice; Molecular Imaging; Neoplasm Transplantation; Promoter Regions, Genetic; alpha-Fetoproteins
PubMed: 27468205
DOI: 10.3748/wjg.v22.i27.6127