scholarly journals NATF (Native And Tissue-specific Fluorescence): A strategy for bright, tissue-specific GFP labeling of native proteins

2018 ◽  
Author(s):  
Siwei He ◽  
Andrea Cuentas-Condori ◽  
David M. Miller
Keyword(s):  
Cell Types ◽  
Specific Protein ◽  
Target Protein ◽  
Specific Cell ◽  
Genomic Locus ◽  
Intracellular Location ◽  
Tissue Specific ◽  
Specific Fluorescence ◽  
C Elegans ◽  
Native Proteins

ABSTRACTGFP labeling by genome editing can reveal the authentic location of a native protein but is frequently hampered by weak GFP signals and broad expression across a range cell types in multicellular animals. To overcome these problems, we engineered a Native And Tissue-specific Fluorescence (NATF) strategy which combines CRISPR/Cas-9 and split-GFP to yield bright, cell-specific protein labeling. We use CRISPR/Cas9 to insert a tandem array of seven copies of the GFP11 β-strand (gfp11x7) at the genomic locus of each target protein. The resultant gfp11x7 knock-in strain is then crossed with separate reporter lines that express the complementing split-GFP fragment (gfp1-10) in specific cell types thus affording tissue-specific labeling of the target protein at its native level. We show that NATF reveals the otherwise undetectable intracellular location of the immunoglobulin protein, OIG-1, and demarcates a receptor auxiliary protein LEV-10 at cell-specific synaptic domains in the C. elegans nervous system.

scholarly journals Tissue specific targeting of DNA nanodevices in a multicellular living organism

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Kasturi Chakraborty ◽  
Palapuravan Anees ◽  
Sunaina Surana ◽  
Simona Martin ◽  
Jihad Aburas ◽  
...  
Keyword(s):  
Living Organism ◽  
Cell Types ◽  
Specific Cell ◽  
Full Potential ◽  
Tissue Specific ◽  
C Elegans ◽  
Specific Targeting ◽  
Synthetic Receptor ◽  
Ligand Interactions

Nucleic acid nanodevices present great potential as agents for logic-based therapeutic intervention as well as in basic biology. Often, however, the disease targets that need corrective action are localized in specific organs and thus realizing the full potential of DNA nanodevices also requires ways to target them to specific cell-types in vivo. Here we show that by exploiting either endogenous or synthetic receptor-ligand interactions and by leveraging the biological barriers presented by the organism, we can target extraneously introduced DNA nanodevices to specific cell types in C. elegans, with sub-cellular precision. The amenability of DNA nanostructures to tissue-specific targeting in vivo significantly expands their utility in biomedical applications and discovery biology.

scholarly journals Tissue specific targeting of DNA nanodevices in a multicellular living organism

2021 ◽  
Author(s):  
Kasturi Chakraborty ◽  
Sunaina Surana ◽  
Simona Martin ◽  
Jihad Aburas ◽  
Sandrine Moutel ◽  
...  
Keyword(s):  
Living Organism ◽  
Cell Types ◽  
Specific Cell ◽  
Full Potential ◽  
Tissue Specific ◽  
C Elegans ◽  
Specific Targeting ◽  
Synthetic Receptor ◽  
Ligand Interactions

AbstractNucleic acid nanodevices present great potential as agents for logic-based therapeutic intervention as well as in basic biology. Often, however, the disease targets that need corrective action are localized in specific organs and thus realizing the full potential of DNA nanodevices also requires ways to target them to specific cell-types in vivo. Here we show that by exploiting either endogenous or synthetic receptor-ligand interactions and by leveraging the biological barriers presented by the organism, we can target extraneously introduced DNA nanodevices to specific cell types in C. elegans, with sub-cellular precision. The amenability of DNA nanostructures to tissue-specific targeting in vivo significantly expands their utility in biomedical applications and discovery biology.

scholarly journals Adult tissue-resident stem cells—fact or fiction?

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Deepa Bhartiya
Keyword(s):  
Stem Cells ◽  
Single Cell ◽  
Adult Stem Cells ◽  
Cell Types ◽  
Specific Cell ◽  
Tissue Specific ◽  
Cardiac Tissues ◽  
Adult Tissues ◽  
Resident Stem Cells ◽  
Abundant Cytoplasm

AbstractLife-long tissue homeostasis of adult tissues is supposedly maintained by the resident stem cells. These stem cells are quiescent in nature and rarely divide to self-renew and give rise to tissue-specific “progenitors” (lineage-restricted and tissue-committed) which divide rapidly and differentiate into tissue-specific cell types. However, it has proved difficult to isolate these quiescent stem cells as a physical entity. Recent single-cell RNAseq studies on several adult tissues including ovary, prostate, and cardiac tissues have not been able to detect stem cells. Thus, it has been postulated that adult cells dedifferentiate to stem-like state to ensure regeneration and can be defined as cells capable to replace lost cells through mitosis. This idea challenges basic paradigm of development biology regarding plasticity that a cell enters point of no return once it initiates differentiation. The underlying reason for this dilemma is that we are putting stem cells and somatic cells together while processing for various studies. Stem cells and adult mature cell types are distinct entities; stem cells are quiescent, small in size, and with minimal organelles whereas the mature cells are metabolically active and have multiple organelles lying in abundant cytoplasm. As a result, they do not pellet down together when centrifuged at 100–350g. At this speed, mature cells get collected but stem cells remain buoyant and can be pelleted by centrifuging at 1000g. Thus, inability to detect stem cells in recently published single-cell RNAseq studies is because the stem cells were unknowingly discarded while processing and were never subjected to RNAseq. This needs to be kept in mind before proposing to redefine adult stem cells.

scholarly journals Chimeric Maternal Cells with Tissue-Specific Antigen Expression and Morphology Are Common in Infant Tissues

2009 ◽  
Vol 12 (5) ◽  
pp. 337-346 ◽  
Author(s):  
Anne M. Stevens ◽  
Heidi M. Hermes ◽  
Meghan M. Kiefer ◽  
Joe C. Rutledge ◽  
J. Lee Nelson
Keyword(s):  
Islet Cells ◽  
Tissue Injury ◽  
Specific Antigen ◽  
Antigen Expression ◽  
Cell Types ◽  
Target Cells ◽  
Specific Cell ◽  
Tissue Specific ◽  
Renal Tubular Cells ◽  
Parenchymal Cells

Maternal microchimerism (MMc) has been purported to play a role in the pathogenesis of autoimmunity, but how a small number of foreign cells could contribute to chronic, systemic inflammation has not been explained. Reports of peripheral blood cells differentiating into tissue-specific cell types may shed light on the problem in that chimeric maternal cells could act as target cells within tissues. We investigated MMc in tissues from 7 male infants. Female cells, presumed maternal, were characterized by simultaneous immunohistochemistry and fluorescence in situ hybridization for X- and Y-chromosomes. Maternal cells constituted 0.017% to 1.9% of parenchymal cells and were found in all infants in liver, pancreas, lung, kidney, bladder, skin, and spleen. Maternal cells were differentiated: maternal hepatocytes in liver, renal tubular cells in kidney, and β-islet cells in pancreas. Maternal cells were not found in areas of tissue injury or inflammatory infiltrate. Maternal hematopoietic cells were found only in hearts from patients with neonatal lupus. Thus, differentiated maternal cells are present in multiple tissue types and occur independently of inflammation or tissue injury. Loss of tolerance to maternal parenchymal cells could lead to organ-specific “auto” inflammatory disease and elimination of maternal cells in areas of inflammation.

scholarly journals Generation of asymmetry during development. Segregation of type-specific proteins in Caulobacter.

1979 ◽  
Vol 81 (1) ◽  
pp. 123-136 ◽  
Author(s):  
N Agabian ◽  
M Evinger ◽  
G Parker
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Messenger Rna ◽  
Cell Types ◽  
Specific Protein ◽  
Soluble Proteins ◽  
Specific Cell ◽  
Daughter Cells ◽  
Specific Proteins ◽  
Entire Cell

An essential event in developmental processes is the introduction of asymmetry into an otherwise undifferentiated cell population. Cell division in Caulobacter is asymmetric; the progeny cells are structurally different and follow different sequences of development, thus providing a useful model system for the study of differentiation. Because the progeny cells are different from one another, there must be a segregation of morphogenetic and informational components at some time in the cell cycle. We have examined the pattern of specific protein segregation between Caulobacter stalked and swarmer daughter cells, with the rationale that such a progeny analysis would identify both structurally and developmentally important proteins. To complement the study, we have also examined the pattern of protein synthesis during synchronous growth and in various cellular fractions. We show here, for the first time, that the association of proteins with a specific cell type may result not only from their periodicity of synthesis, but also from their pattern of distribution at the time of cell division. Several membrane-associated and soluble proteins are segregated asymmetrically between progeny stalked and swarmer cells. The data further show that a subclass of soluble proteins becomes associated with the membrane of the progeny stalked cells. Therefore, although the principal differentiated cell types possess different synthetic capabilities and characteristic proteins, the asymmetry between progeny stalked and swarmer cells is generated primarily by the preferential association of specific soluble proteins with the membrane of only one daughter cell. The majority of the proteins which exhibit this segregation behavior are synthesized during the entire cell cycle and exhibit relatively long, functional messenger RNA half-lives.

scholarly journals Tissue-Specific Transcription Footprinting Using RNA PoI DamID (RAPID) in Caenorhabditis elegans

Genetics ◽  
2020 ◽  
Vol 216 (4) ◽  
pp. 931-945 ◽  
Author(s):  
Georgina Gómez-Saldivar ◽  
Jaime Osuna-Luque ◽  
Jennifer I. Semple ◽  
Dominique A. Glauser ◽  
Sophie Jarriault ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Rna Polymerase ◽  
Cell Types ◽  
Neuroendocrine Cells ◽  
Cell Physiology ◽  
Specific Gene ◽  
Cell Content ◽  
Tissue Specific ◽  
C Elegans ◽  
Active Transcription

Differential gene expression across cell types underlies development and cell physiology in multicellular organisms. Caenorhabditis elegans is a powerful, extensively used model to address these biological questions. A remaining bottleneck relates to the difficulty to obtain comprehensive tissue-specific gene transcription data, since available methods are still challenging to execute and/or require large worm populations. Here, we introduce the RNA Polymerase DamID (RAPID) approach, in which the Dam methyltransferase is fused to a ubiquitous RNA polymerase subunit to create transcriptional footprints via methyl marks on the DNA of transcribed genes. To validate the method, we determined the polymerase footprints in whole animals, in sorted embryonic blastomeres and in different tissues from intact young adults by driving tissue-specific Dam fusion expression. We obtained meaningful transcriptional footprints in line with RNA-sequencing (RNA-seq) studies in whole animals or specific tissues. To challenge the sensitivity of RAPID and demonstrate its utility to determine novel tissue-specific transcriptional profiles, we determined the transcriptional footprints of the pair of XXX neuroendocrine cells, representing 0.2% of the somatic cell content of the animals. We identified 3901 candidate genes with putatively active transcription in XXX cells, including the few previously known markers for these cells. Using transcriptional reporters for a subset of new hits, we confirmed that the majority of them were expressed in XXX cells and identified novel XXX-specific markers. Taken together, our work establishes RAPID as a valid method for the determination of RNA polymerase footprints in specific tissues of C. elegans without the need for cell sorting or RNA tagging.

scholarly journals Inducible Systemic RNA Silencing in Caenorhabditis elegans

2003 ◽  
Vol 14 (7) ◽  
pp. 2972-2983 ◽  
Author(s):  
Lisa Timmons ◽  
Hiroaki Tabara ◽  
Craig C. Mello ◽  
Andrew Z. Fire
Keyword(s):  
Rna Interference ◽  
Caenorhabditis Elegans ◽  
Rna Silencing ◽  
Molecular Mechanisms ◽  
Normal Animal ◽  
Cell Types ◽  
Animal Cells ◽  
Tissue Specific ◽  
C Elegans ◽  
Systemic Rna

Introduction of double-stranded RNA (dsRNA) can elicit a gene-specific RNA interference response in a variety of organisms and cell types. In many cases, this response has a systemic character in that silencing of gene expression is observed in cells distal from the site of dsRNA delivery. The molecular mechanisms underlying the mobile nature of RNA silencing are unknown. For example, although cellular entry of dsRNA is possible, cellular exit of dsRNA from normal animal cells has not been directly observed. We provide evidence that transgenic strains of Caenorhabditis elegans transcribing dsRNA from a tissue-specific promoter do not exhibit comprehensive systemic RNA interference phenotypes. In these same animals, modifications of environmental conditions can result in more robust systemic RNA silencing. Additionally, we find that genetic mutations can influence the systemic character of RNA silencing in C. elegans and can separate mechanisms underlying systemic RNA silencing into tissue-specific components. These data suggest that trafficking of RNA silencing signals in C. elegans is regulated by specific physiological and genetic factors.

scholarly journals SCISSOR™: a single-cell inferred site-specific omics resource for tumor microenvironment association study

NAR Cancer ◽  
2021 ◽  
Vol 3 (3) ◽  
Author(s):  
Xiang Cui ◽  
Fei Qin ◽  
Xuanxuan Yu ◽  
Feifei Xiao ◽  
Guoshuai Cai
Keyword(s):  
Tumor Microenvironment ◽  
Single Cell ◽  
Clinical Outcomes ◽  
Large Scale ◽  
Cell Types ◽  
Cell Interaction ◽  
Specific Cell ◽  
Dynamic Visualization ◽  
Tissue Specific ◽  
Cell Composition

Abstract Tumor tissues are heterogeneous with different cell types in tumor microenvironment, which play an important role in tumorigenesis and tumor progression. Several computational algorithms and tools have been developed to infer the cell composition from bulk transcriptome profiles. However, they ignore the tissue specificity and thus a new resource for tissue-specific cell transcriptomic reference is needed for inferring cell composition in tumor microenvironment and exploring their association with clinical outcomes and tumor omics. In this study, we developed SCISSOR™ (https://thecailab.com/scissor/), an online open resource to fulfill that demand by integrating five orthogonal omics data of >6031 large-scale bulk samples, patient clinical outcomes and 451 917 high-granularity tissue-specific single-cell transcriptomic profiles of 16 cancer types. SCISSOR™ provides five major analysis modules that enable flexible modeling with adjustable parameters and dynamic visualization approaches. SCISSOR™ is valuable as a new resource for promoting tumor heterogeneity and tumor–tumor microenvironment cell interaction research, by delineating cells in the tissue-specific tumor microenvironment and characterizing their associations with tumor omics and clinical outcomes.

scholarly journals Comprehensive characterization of tissue-specific chromatin accessibility in L2 Caenorhabditis elegans nematodes

2020 ◽  
Author(s):  
Timothy J. Durham ◽  
Riza M. Daza ◽  
Louis Gevirtzman ◽  
Darren A. Cusanovich ◽  
William Stafford Noble ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Expression Patterns ◽  
Cell Types ◽  
Chromatin Accessibility ◽  
Gene Expression Patterns ◽  
Rna Seq ◽  
Cell Type ◽  
Tissue Specific ◽  
C Elegans

AbstractRecently developed single cell technologies allow researchers to characterize cell states at ever greater resolution and scale. C. elegans is a particularly tractable system for studying development, and recent single cell RNA-seq studies characterized the gene expression patterns for nearly every cell type in the embryo and at the second larval stage (L2). Gene expression patterns are useful for learning about gene function and give insight into the biochemical state of different cell types; however, in order to understand these cell types, we must also determine how these gene expression levels are regulated. We present the first single cell ATAC-seq study in C. elegans. We collected data in L2 larvae to match the available single cell RNA-seq data set, and we identify tissue-specific chromatin accessibility patterns that align well with existing data, including the L2 single cell RNA-seq results. Using a novel implementation of the latent Dirichlet allocation algorithm, we leverage the single-cell resolution of the sci-ATAC-seq data to identify accessible loci at the level of individual cell types, providing new maps of putative cell type-specific gene regulatory sites, with promise for better understanding of cellular differentiation and gene regulation in the worm.

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