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Kidney International Dec 2021Lineage tracing was originally developed by developmental biologists to identify all progeny of a single cell during morphogenesis. More recently this approach has been... (Review)
Review
Lineage tracing was originally developed by developmental biologists to identify all progeny of a single cell during morphogenesis. More recently this approach has been applied to other fields, including organ homeostasis and recovery from injury. Modern lineage tracing techniques typically rely on reporter gene expression induced by cell-specific DNA recombination. There have been important scientific advances in the last 10 years that have impacted lineage tracing approaches, including intersectional genetics, optical clearing techniques, and the use of sequencing-based genomic lineage tracing. The latter combines CRISPR-Cas9-based genetic scarring with single-cell RNA-sequencing that, in theory, could allow comprehensive reconstruction of a lineage tree for an entire organism. This review summarizes recent advances in lineage tracing technologies and outlines potential applications for kidney research.
Topics: CRISPR-Cas Systems; Cell Lineage; Gene Expression; Genes, Reporter; Genomics; Humans; Kidney
PubMed: 34217781
DOI: 10.1016/j.kint.2021.05.040 -
International Journal of Molecular... Nov 2022Tumor suppressor protein P53 induces cycle arrest and apoptosis by mediating the transcriptional expression of its target genes. Mutations causing conformational...
Tumor suppressor protein P53 induces cycle arrest and apoptosis by mediating the transcriptional expression of its target genes. Mutations causing conformational abnormalities and post-translational modifications that promote degradation are the main reasons for the loss of P53 function in tumor cells. Reporter gene assays that can scientifically reflect the biological function can help discover the mechanism and therapeutic strategies that restore P53 function. In the reporter gene system of this work, tetracycline-inducible expression of wild-type was used to provide a fully activated state as a 100% activity reference for the objective measurement of biological function. It was confirmed by RT-qPCR, cell viability assay, immunofluorescence, and Western blot analysis that the above-mentioned reporter gene system could correctly reflect the differences in biological activity between the wild-type and mutants. After that, the system was tentatively used for related mechanism research and compound activity evaluation. Through the tetracycline-induced co-expression of wild-type and mutant in exact proportion, it was observed that the response modes of typical transcriptional response elements (TREs) to dominant negative mutation effect were not exactly the same. Compared to the relative multiple-to-solvent control, the activity percentage relative to the 100% activity reference of wild-type can better reflect the actual influence of the so-called P53 mutant reactivator. Similarly, relative to the 100% activity reference, it can objectively reflect the biological effects caused by the inhibitor of negative factors, such as MDM2. In conclusion, this study provides a 100% activity reference and a reliable calculation model for relevant basic research and drug development.
Topics: Tumor Suppressor Protein p53; Genes, Reporter; Mutation; Response Elements; Tetracyclines
PubMed: 36430341
DOI: 10.3390/ijms232213867 -
Molecular Imaging and Biology Jun 2017Biomedical synthetic biology is an emerging field in which cells are engineered at the genetic level to carry out novel functions with relevance to biomedical and... (Review)
Review
Biomedical synthetic biology is an emerging field in which cells are engineered at the genetic level to carry out novel functions with relevance to biomedical and industrial applications. This approach promises new treatments, imaging tools, and diagnostics for diseases ranging from gastrointestinal inflammatory syndromes to cancer, diabetes, and neurodegeneration. As these cellular technologies undergo pre-clinical and clinical development, it is becoming essential to monitor their location and function in vivo, necessitating appropriate molecular imaging strategies, and therefore, we have created an interest group within the World Molecular Imaging Society focusing on synthetic biology and reporter gene technologies. Here, we highlight recent advances in biomedical synthetic biology, including bacterial therapy, immunotherapy, and regenerative medicine. We then discuss emerging molecular imaging approaches to facilitate in vivo applications, focusing on reporter genes for noninvasive modalities such as magnetic resonance, ultrasound, photoacoustic imaging, bioluminescence, and radionuclear imaging. Because reporter genes can be incorporated directly into engineered genetic circuits, they are particularly well suited to imaging synthetic biological constructs, and developing them provides opportunities for creative molecular and genetic engineering.
Topics: Animals; Cell Engineering; Genes, Reporter; Genetic Vectors; Humans; Molecular Imaging; Synthetic Biology
PubMed: 28213833
DOI: 10.1007/s11307-017-1062-1 -
Genome Research Oct 2019The cleavage and polyadenylation reaction is a crucial step in transcription termination and pre-mRNA maturation in human cells. Despite extensive research, the encoding...
The cleavage and polyadenylation reaction is a crucial step in transcription termination and pre-mRNA maturation in human cells. Despite extensive research, the encoding of polyadenylation-mediated regulation of gene expression within the DNA sequence is not well understood. Here, we utilized a massively parallel reporter assay to inspect the effect of over 12,000 rationally designed polyadenylation sequences (PASs) on reporter gene expression and cleavage efficiency. We find that the PAS sequence can modulate gene expression by over five orders of magnitude. By using a uniquely designed scanning mutagenesis data set, we gain mechanistic insight into various modes of action by which the cleavage efficiency affects the sensitivity or robustness of the PAS to mutation. Furthermore, we employ motif discovery to identify both known and novel sequence motifs associated with PAS-mediated regulation. By leveraging the large scale of our data, we train a deep learning model for the highly accurate prediction of RNA levels from DNA sequence alone ( = 0.83). Moreover, we devise unique approaches for predicting exact cleavage sites for our reporter constructs and for endogenous transcripts. Taken together, our results expand our understanding of PAS-mediated regulation, and provide an unprecedented resource for analyzing and predicting PAS for regulatory genomics applications.
Topics: 3' Untranslated Regions; Deep Learning; Gene Expression Regulation; Genes, Reporter; Genomics; High-Throughput Nucleotide Sequencing; Humans; Mutagenesis; Polyadenylation; RNA Precursors; RNA, Messenger; Transcription Termination, Genetic
PubMed: 31530582
DOI: 10.1101/gr.247312.118 -
NMR in Biomedicine Jul 2013Molecular imaging strives to detect molecular events at the level of the whole organism. In some cases, the molecule of interest can be detected either directly or with... (Review)
Review
Molecular imaging strives to detect molecular events at the level of the whole organism. In some cases, the molecule of interest can be detected either directly or with targeted contrast media. However many genes and proteins and particularly those located in intracellular compartments are not accessible for targeted agents. The transcriptional regulation of these genes can nevertheless be detected, although indirectly, using reporter gene encoding for readily detectable proteins. Such reporter proteins can be expressed in the tissue of interest by genetically introducing the reporter gene in the target cells. Imaging of reporter genes has become a powerful tool in modern biomedical research. Typically, expression of fluorescent and bioluminescent proteins and the reaction product of expressed enzymes and exogenous substrates were examined using in vitro histological methods and in vivo whole body imaging methods. Recent advances in MRI reporter gene methods raised the possibility that MRI could become a powerful tool for concomitant high-resolution anatomical and functional imaging and for imaging of reporter gene activity. An immediate application of MRI reporter gene methods was by monitoring gene expression patterns in gene therapy and in vivo imaging of the survival, proliferation, migration and differentiation of pluripotent and multipotent cells used in cell-based regenerative therapies for cancer, myocardial infarction and neural degeneration. In this review, we characterized a variety of MRI reporter gene methods based on their applicability to report cell survival/proliferation, migration and differentiation. In particular, we discussed which methods were best suited for translation to clinical use in regenerative therapies.
Topics: Animals; Cell Differentiation; Cell Movement; Cell Proliferation; Cell Survival; Genes, Reporter; Humans; Magnetic Resonance Imaging
PubMed: 23225197
DOI: 10.1002/nbm.2869 -
BioTechniques Dec 2023Fluorescent proteins, such as green fluorescent proteins, are invaluable tools for detecting and quantifying gene expression in high-throughput reporter gene assays....
Fluorescent proteins, such as green fluorescent proteins, are invaluable tools for detecting and quantifying gene expression in high-throughput reporter gene assays. However, they introduce significant inaccuracies in studies involving microaerobiosis or anaerobiosis, as oxygen is required for the maturation of these proteins' chromophores. In this study, the authors highlight the errors incurred by using fluorescent proteins under limited oxygenation by comparing standard fluorescence-based reporter gene assays to quantitative real-time PCR data in the study of a complex oxygen-regulated gene network. Furthermore, a solution to perform quantification of anaerobic and microaerobic gene expression with fluorescent reporter proteins using a microplate reader with an oxygen control system and applying pulses of full oxygenation before fluorescence measurements is provided.
Topics: Anaerobiosis; Genes, Reporter; Green Fluorescent Proteins; Indicators and Reagents; Gene Expression; Oxygen; Luminescent Proteins
PubMed: 37880975
DOI: 10.2144/btn-2023-0064 -
Trends in Genetics : TIG Oct 2022MRI-based gene reporters allow imaging of gene expression at depth (tens of centimetres) and at relatively high resolution (~10-100 μm) and have the potential to be...
MRI-based gene reporters allow imaging of gene expression at depth (tens of centimetres) and at relatively high resolution (~10-100 μm) and have the potential to be translated to the clinic. The reporters exploit either endogenous contrast mechanisms or they modulate the response to an introduced exogenous contrast agent.
Topics: Genes, Reporter; Image Enhancement; Magnetic Resonance Imaging
PubMed: 35641343
DOI: 10.1016/j.tig.2022.05.006 -
Genome Biology Feb 2023A long-standing goal of evolutionary biology is to decode how gene regulation contributes to organismal diversity. Doing so is challenging because it is hard to predict... (Review)
Review
A long-standing goal of evolutionary biology is to decode how gene regulation contributes to organismal diversity. Doing so is challenging because it is hard to predict function from non-coding sequence and to perform molecular research with non-model taxa. Massively parallel reporter assays (MPRAs) enable the testing of thousands to millions of sequences for regulatory activity simultaneously. Here, we discuss the execution, advantages, and limitations of MPRAs, with a focus on evolutionary questions. We propose solutions for extending MPRAs to rare taxa and those with limited genomic resources, and we underscore MPRA's broad potential for driving genome-scale, functional studies across organisms.
Topics: Sequence Analysis, DNA; Gene Expression Regulation; Genomics; Genes, Reporter
PubMed: 36788564
DOI: 10.1186/s13059-023-02856-6 -
Theranostics 2016Photoacoustic (PA) imaging is a rapidly emerging biomedical imaging modality that is capable of visualizing cellular and molecular functions with high detection... (Review)
Review
Photoacoustic (PA) imaging is a rapidly emerging biomedical imaging modality that is capable of visualizing cellular and molecular functions with high detection sensitivity and spatial resolution in deep tissue. Great efforts and progress have been made on the development of various PA imaging technologies with improved resolution and sensitivity over the past two decades. Various PA probes with high contrast have also been extensively developed, with many important biomedical applications. In comparison with chemical dyes and nanoparticles, genetically encoded probes offer easier labeling of defined cells within tissues or proteins of interest within a cell, have higher stability vo, and eliminate the need for delivery of exogenous substances. Genetically encoded probes have thus attracted increasing attention from researchers in engineering and biomedicine. In this review, we aim to provide an overview of the existing PA imaging technologies and genetically encoded PA probes, and describe further improvements in PA imaging techniques and the near-infrared photochromic protein BphP1, the most sensitive genetically encoded probe thus far, as well as the potential biomedical applications of BphP1-based PA imaging .
Topics: Animals; Genes, Reporter; Humans; Molecular Imaging; Optical Imaging; Photoacoustic Techniques
PubMed: 27877244
DOI: 10.7150/thno.15878 -
Current Opinion in Biotechnology Feb 2007In the past few years there has been a veritable explosion in the field of reporter gene imaging, with the aim of determining the location, duration and extent of gene... (Review)
Review
In the past few years there has been a veritable explosion in the field of reporter gene imaging, with the aim of determining the location, duration and extent of gene expression within living subjects. An important application of this approach is the molecular imaging of interacting protein partners, which could pave the way to functional proteomics in living animals and might provide a tool for the whole-body evaluation of new pharmaceuticals targeted to modulate protein-protein interactions. Three general methods are currently available for imaging protein-protein interactions in living subjects using reporter genes: a modified mammalian two-hybrid system, a bioluminescence resonance energy transfer (BRET) system, and split reporter protein complementation and reconstitution strategies. In the future, these innovative approaches are likely to enhance our appreciation of entire biological pathway systems and their pharmacological regulation.
Topics: Animals; Gene Expression; Genes, Reporter; Luciferases; Luminescent Measurements; Proteins
PubMed: 17254764
DOI: 10.1016/j.copbio.2007.01.007