scholarly journals CRISPR-TSKO facilitates efficient cell type-, tissue-, or organ-specific mutagenesis in Arabidopsis

2018 ◽  
Author(s):  
Ward Decaestecker ◽  
Rafael Andrade Buono ◽  
Marie L. Pfeiffer ◽  
Nick Vangheluwe ◽  
Joris Jourquin ◽  
...  
Keyword(s):  
Lateral Roots ◽  
Cell Types ◽  
Loss Of Function ◽  
Cellular Functions ◽  
Functional Studies ◽  
Organ Specific ◽  
Plant Genes ◽  
Mutant Lines ◽  
Functional Analyses ◽  
Efficient Selection

AbstractDetailed functional analyses of many fundamentally-important plant genes via conventional loss-of-function approaches are impeded by severe pleiotropic phenotypes. In particular, mutations in genes that are required for basic cellular functions and/or reproduction often interfere with the generation of homozygous mutant plants, precluding further functional studies. To overcome this limitation, we devised a CRISPR-based tissue-specific knockout system, CRISPR-TSKO, enabling the generation of somatic mutations in particular plant cell types, tissues, and organs. In Arabidopsis, CRISPR-TSKO mutations in essential genes caused well-defined, localized phenotypes in the root cap, stomatal lineage, or entire lateral roots. The underlying modular cloning system allows for efficient selection, identification, and functional analysis of mutant lines directly in the first transgenic generation. The efficacy of CRISPR-TSKO opens new avenues to discover and analyze gene functions in spatial and temporal contexts of plant life while avoiding pleiotropic effects of system-wide loss of gene function.

par-4, a gene required for cytoplasmic localization and determination of specific cell types in Caenorhabditis elegans embryogenesis.

Genetics ◽  
1992 ◽  
Vol 130 (4) ◽  
pp. 771-790 ◽  
Author(s):  
D G Morton ◽  
J M Roos ◽  
K J Kemphues
Keyword(s):  
Caenorhabditis Elegans ◽  
Cell Types ◽  
Intestinal Cells ◽  
Specific Cell ◽  
Cytoplasmic Localization ◽  
Loss Of Function ◽  
Embryo Viability ◽  
Cell Stage ◽  
Maternal Genome

Abstract Specification of some cell fates in the early Caenorhabditis elegans embryo is mediated by cytoplasmic localization under control of the maternal genome. Using nine newly isolated mutations, and two existing mutations, we have analyzed the role of the maternally expressed gene par-4 in cytoplasmic localization. We recovered seven new par-4 alleles in screens for maternal effect lethal mutations that result in failure to differentiate intestinal cells. Two additional par-4 mutations were identified in noncomplementation screens using strains with a high frequency of transposon mobility. All 11 mutations cause defects early in development of embryos produced by homozygous mutant mothers. Analysis with a deficiency in the region indicates that it33 is a strong loss-of-function mutation. par-4(it33) terminal stage embryos contain many cells, but show no morphogenesis, and are lacking intestinal cells. Temperature shifts with the it57ts allele suggest that the critical period for both intestinal differentiation and embryo viability begins during oogenesis, about 1.5 hr before fertilization, and ends before the four-cell stage. We propose that the primary function of the par-4 gene is to act as part of a maternally encoded system for cytoplasmic localization in the first cell cycle, with par-4 playing a particularly important role in the determination of intestine. Analysis of a par-4; par-2 double mutant suggests that par-4 and par-2 gene products interact in this system.

scholarly journals Metabolic activation and colitis pathogenesis is prevented by lymphotoxin β receptor expression in neutrophils

2021 ◽  
Author(s):  
Thomas Riffelmacher ◽  
Daniel A. Giles ◽  
Sonja Zahner ◽  
Martina Dicker ◽  
Alexander Y. Andreyev ◽  
...  
Keyword(s):  
Severe Disease ◽  
Metabolic Activation ◽  
Cell Types ◽  
Receptor Expression ◽  
Neutrophil Accumulation ◽  
Functional Studies ◽  
Mitochondrial Ros ◽  
Tnf Superfamily ◽  
Β Receptor ◽  
Lymphotoxin Beta Receptor

AbstractInflammatory bowel disease is characterized by an exacerbated intestinal immune response, but the critical mechanisms regulating immune activation remain incompletely understood. We previously reported that the TNF-superfamily molecule TNFSF14 (LIGHT) is required for preventing severe disease in mouse models of colitis. In addition, deletion of lymphotoxin beta receptor (LTβR), which binds LIGHT, also led to aggravated colitis pathogenesis. Here, we aimed to determine the cell type(s) requiring LTβR and the mechanism critical for exacerbation of colitis. Specific deletion of LTβR in neutrophils (LTβRΔN), but not in several other cell types, was sufficient to induce aggravated colitis and colonic neutrophil accumulation. Mechanistically, RNA-Seq analysis revealed LIGHT-induced suppression of cellular metabolism, and mitochondrial function, that was dependent on LTβR. Functional studies confirmed increased mitochondrial mass and activity, associated with excessive mitochondrial ROS production and elevated glycolysis at steady-state and during colitis. Targeting these metabolic changes rescued exacerbated disease severity. Our results demonstrate that LIGHT signals to LTβR on neutrophils to suppress metabolic activation and thereby prevents exacerbated immune pathogenesis during colitis.

scholarly journals Roles for RNA export factor, Nxt1, in ensuring muscle integrity and normal RNA expression in Drosophila

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Kevin van der Graaf ◽  
Katia Jindrich ◽  
Robert Mitchell ◽  
Helen White-Cooper
Keyword(s):  
Mrna Export ◽  
Rna Stability ◽  
Muscle Degeneration ◽  
Loss Of Function ◽  
Cellular Functions ◽  
Partial Loss ◽  
Export Pathway ◽  
Pathway Gene ◽  
Rna Export

Abstract The mRNA export pathway is responsible for the transport of mRNAs from the nucleus to the cytoplasm, and thus is essential for protein production and normal cellular functions. A partial loss of function allele of the mRNA export factor Nxt1 in Drosophila shows reduced viability and sterility. A previous study has shown that the male fertility defect is due to a defect in transcription and RNA stability, indicating the potential for this pathway to be implicated in processes beyond the known mRNA transport function. Here we investigate the reduced viability of Nxt1 partial loss of function mutants, and describe a defect in growth and maintenance of the larval muscles, leading to muscle degeneration. RNA-seq revealed reduced expression of a set of mRNAs, particularly from genes with long introns in Nxt1 mutant carcass. We detected differential expression of circRNA, and significantly fewer distinct circRNAs expressed in the mutants. Despite the widespread defects in gene expression, muscle degeneration was rescued by increased expression of the costamere component tn (abba) in muscles. This is the first report of a role for the RNA export pathway gene Nxt1 in the maintenance of muscle integrity. Our data also links the mRNA export pathway to a specific role in the expression of mRNA and circRNA from common precursor genes, in vivo.

scholarly journals miR-16-5p Promotes Erythroid Maturation of Erythroleukemia Cells by Regulating Ribosome Biogenesis

2021 ◽  
Vol 14 (2) ◽  
pp. 137
Author(s):  
Christos I. Papagiannopoulos ◽  
Nikoleta F. Theodoroula ◽  
Ioannis S. Vizirianakis
Keyword(s):  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Cultured Cells ◽  
Erythroid Differentiation ◽  
Underlying Mechanism ◽  
Loss Of Function ◽  
Functional Studies ◽  
Differentiated Cells ◽  
Erythroleukemia Cells ◽  
Murine Erythroleukemia

miRNAs constitute a class of non-coding RNA that act as powerful epigenetic regulators in animal and plant cells. In order to identify putative tumor-suppressor miRNAs we profiled the expression of various miRNAs during differentiation of erythroleukemia cells. RNA was purified before and after differentiation induction and subjected to quantitative RT-PCR. The majority of the miRNAs tested were found upregulated in differentiated cells with miR-16-5p showing the most significant increase. Functional studies using gain- and loss-of-function constructs proposed that miR-16-5p has a role in promoting the erythroid differentiation program of murine erythroleukemia (MEL) cells. In order to identify the underlying mechanism of action, we utilized bioinformatic in-silico platforms that incorporate predictions for the genes targeted by miR-16-5p. Interestingly, ribosome constituents, as well as ribosome biogenesis factors, were overrepresented among the miR-16-5p predicted gene targets. Accordingly, biochemical experiments showed that, indeed, miR-16-5p could modulate the levels of independent ribosomal proteins, and the overall ribosomal levels in cultured cells. In conclusion, miR-16-5p is identified as a differentiation-promoting agent in erythroleukemia cells, demonstrating antiproliferative activity, likely as a result of its ability to target the ribosomal machinery and restore any imbalanced activity imposed by the malignancy and the blockade of differentiation.

scholarly journals Genetic Vectors as a Tool in Association Studies: Definitions and Application for Study of Rheumatoid Arthritis

2015 ◽  
Vol 2015 ◽  
pp. 1-13
Author(s):  
Igor Sandalov ◽  
Leonid Padyukov
Keyword(s):  
Rheumatoid Arthritis ◽  
Complex Disease ◽  
Association Studies ◽  
Healthy Population ◽  
Common Complex Disease ◽  
New Approach ◽  
Functional Studies ◽  
Biological Interpretation ◽  
Genetic Vectors ◽  
Efficient Selection

To identify putative relations between different genetic factors in the human genome in the development of common complex disease, we mapped the genetic data to an ensemble of spin chains and analysed the data as a quantum system. Each SNP is considered as a spin with three states corresponding to possible genotypes. The combined genotype represents a multispin state, described by the product of individual-spin states. Each person is characterized by a single genetic vector (GV) and individuals with identical GVs comprise the GV group. This consolidation of genotypes into GVs provides integration of multiple genetic variants for a single statistical test and excludes ambiguity of biological interpretation known for allele and haplotype associations. We analyzed two independent cohorts, with 2633 rheumatoid arthritis cases and 2108 healthy controls, and data for 6 SNPs from the HTR2A locus plus shared epitope allele. We found that GVs based on selected markers are highly informative and overlap for 98.3% of the healthy population between two cohorts. Interestingly, some of the GV groups contain either only controls or only cases, thus demonstrating extreme susceptibility or protection features. By using this new approach we confirmed previously detected univariate associations and demonstrated the most efficient selection of SNPs for combined analyses for functional studies.

Stable transfer and restricted expression of a cloned class I gene encoding a secreted transplantation-like antigen

1985 ◽  
Vol 5 (6) ◽  
pp. 1295-1300
Author(s):  
Y Barra ◽  
K Tanaka ◽  
K J Isselbacher ◽  
G Khoury ◽  
G Jay
Keyword(s):  
Molecular Mechanisms ◽  
Cell Types ◽  
Class I ◽  
Regulatory Sequences ◽  
Transcriptional Enhancer ◽  
Gene Encoding ◽  
Specific Expression ◽  
Tissue Specific ◽  
Functional Studies ◽  
I Gene

The identification of a unique major histocompatibility complex class I gene, designated Q10, which encodes a secreted rather than a cell surface antigen has led to questions regarding its potential role in regulating immunological functions. Since the Q10 gene is specifically activated only in the liver, we sought to define the molecular mechanisms which control its expression in a tissue-specific fashion. Results obtained by transfection of the cloned Q10 gene, either in the absence or presence of a heterologous transcriptional enhancer, into a variety of cell types of different tissue derivations are consistent with the Q10 gene being regulated at two levels. The first is by a cis-dependent mechanism which appears to involve site-specific DNA methylation. The second is by a trans-acting mechanism which would include the possibility of an enhancer binding factor. The ability to efficiently express the Q10 gene in certain transfected cell lines offers an opportunity to obtain this secreted class I antigen in quantities sufficient for functional studies; this should also make it possible to define regulatory sequences which may be responsible for the tissue-specific expression of Q10.

Protein kinase C-α and the regulation of diverse cell responses

2017 ◽  
Vol 8 (3-4) ◽  
pp. 143-153 ◽  
Author(s):  
Rishi Kant Singh ◽  
Sanjay Kumar ◽  
Pramod Kumar Gautam ◽  
Munendra Singh Tomar ◽  
Praveen Kumar Verma ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cellular Localization ◽  
Cellular Transformation ◽  
Cell Types ◽  
Adapter Proteins ◽  
Cellular Functions ◽  
Cell Responses ◽  
Kinase C ◽  
Cellular Compartments

AbstractProtein kinase C (PKC) comprises a family of lipid-sensitive enzymes that have been involved in a broad range of cellular functions. PKC-α is a member of classical PKC with ubiquitous expression and different cellular localization. This unique PKC isoform is activated by various signals which evoke lipid hydrolysis, after activation it interacts with various adapter proteins and is localized to specific cellular compartments where it is devised to work. The universal expression and activation by various stimuli make it a perfect player in uncountable cellular functions including differentiation, proliferation, apoptosis, cellular transformation, motility, adhesion and so on. However, these functions are not intrinsic properties of PKC-α, but depend on cell types and conditions. The activities of PKC-α are managed by the various pharmacological activators/inhibitors and antisense oligonucleotides. The aim of this review is to elaborate the structural feature, and provide an insight into the mechanism of PKC-α activation and regulation of its key biological functions in different cellular compartments to develop an effective pharmacological approach to regulate the PKC-α signal array.

scholarly journals Assessment of phagocytic activity in live macrophages-tumor cells co-cultures by Confocal and Nomarski Microscopy

2017 ◽  
Vol 2 (1) ◽  
Author(s):  
Dalia Martinez-Marin ◽  
Courtney Jarvis ◽  
Thomas Nelius ◽  
Stéphanie Filleur
Keyword(s):  
Tumor Microenvironment ◽  
Tumor Cells ◽  
Phagocytic Activity ◽  
Molecular Mechanisms ◽  
Cell Types ◽  
Functional Assay ◽  
Loss Of Function ◽  
Multiple Cancer

Abstract Macrophages have been recognized as the main inflammatory component of the tumor microenvironment. Although often considered as beneficial for tumor growth and disease progression, tumor-associated macrophages have also been shown to be detrimental to the tumor depending on the tumor microenvironment. Therefore, understanding the molecular interactions between macrophages and tumor cells in relation to macrophages functional activities such as phagocytosis is critical for a better comprehension of their tumor-modulating action. Still, the characterization of these molecular mechanisms in vivo remains complicated due to the extraordinary complexity of the tumor microenvironment and the broad range of tumor-associated macrophage functions. Thus, there is an increasing demand for in vitro methodologies to study the role of cell–cell interactions in the tumor microenvironment. In the present study, we have developed live co-cultures of macrophages and human prostate tumor cells to assess the phagocytic activity of macrophages using a combination of Confocal and Nomarski Microscopy. Using this model, we have emphasized that this is a sensitive, measurable, and highly reproducible functional assay. We have also highlighted that this assay can be applied to multiple cancer cell types and used as a selection tool for a variety of different types of phagocytosis agonists. Finally, combining with other studies such as gain/loss of function or signaling studies remains possible. A better understanding of the interactions between tumor cells and macrophages may lead to the identification of new therapeutic targets against cancer.

scholarly journals Transcriptional Changes in Regulatory T Cells From Patients With Autoimmune Polyendocrine Syndrome Type 1 Suggest Functional Impairment of Lipid Metabolism and Gut Homing

2021 ◽  
Vol 12 ◽  
Author(s):  
Amund Holte Berger ◽  
Eirik Bratland ◽  
Thea Sjøgren ◽  
Marte Heimli ◽  
Torgeir Tyssedal ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Type I ◽  
Syndrome Type ◽  
Functional Studies ◽  
Autoimmune Polyendocrine Syndrome ◽  
Organ Specific ◽  
Hla Dqa1 ◽  
And Function ◽  
The Impact

Autoimmune polyendocrine syndrome type I (APS-1) is a monogenic model disorder of organ-specific autoimmunity caused by mutations in the Autoimmune regulator (AIRE) gene. AIRE facilitates the expression of organ-specific transcripts in the thymus, which is essential for efficient removal of dangerous self-reacting T cells and for inducing regulatory T cells (Tregs). Although reduced numbers and function of Tregs have been reported in APS-I patients, the impact of AIRE deficiency on gene expression in these cells is unknown. Here, we report for the first time on global transcriptional patterns of isolated Tregs from APS-1 patients compared to healthy subjects. Overall, we found few differences between the groups, although deviant expression was observed for the genes TMEM39B, SKIDA1, TLN2, GPR15, FASN, BCAR1, HLA-DQA1, HLA-DQB1, HLA-DRA, GPSM3 and AKR1C3. Of significant interest, the consistent downregulation of GPR15 may indicate failure of Treg gut homing which could be of relevance for the gastrointestinal manifestations commonly seen in APS-1. Upregulated FASN expression in APS-1 Tregs points to increased metabolic activity suggesting a putative link to faulty Treg function. Functional studies are needed to determine the significance of these findings for the immunopathogenesis of APS-1 and for Treg immunobiology in general.

scholarly journals A nervous system-specific subnuclear organelle in Caenorhabditis elegans

Genetics ◽  
2021 ◽  
Vol 217 (1) ◽  
Author(s):  
Kenneth Pham ◽  
Neda Masoudi ◽  
Eduardo Leyva-Díaz ◽  
Oliver Hobert
Keyword(s):  
Nervous System ◽  
Caenorhabditis Elegans ◽  
Homeobox Gene ◽  
Cell Types ◽  
Nuclear Bodies ◽  
Loss Of Function ◽  
Cell Type ◽  
Cell Type Specificity ◽  
Splicing Speckles ◽  
Polycomb Bodies

Abstract We describe here phase-separated subnuclear organelles in the nematode Caenorhabditis elegans, which we term NUN (NUclear Nervous system-specific) bodies. Unlike other previously described subnuclear organelles, NUN bodies are highly cell type specific. In fully mature animals, 4–10 NUN bodies are observed exclusively in the nucleus of neuronal, glial and neuron-like cells, but not in other somatic cell types. Based on co-localization and genetic loss of function studies, NUN bodies are not related to other previously described subnuclear organelles, such as nucleoli, splicing speckles, paraspeckles, Polycomb bodies, promyelocytic leukemia bodies, gems, stress-induced nuclear bodies, or clastosomes. NUN bodies form immediately after cell cycle exit, before other signs of overt neuronal differentiation and are unaffected by the genetic elimination of transcription factors that control many other aspects of neuronal identity. In one unusual neuron class, the canal-associated neurons, NUN bodies remodel during larval development, and this remodeling depends on the Prd-type homeobox gene ceh-10. In conclusion, we have characterized here a novel subnuclear organelle whose cell type specificity poses the intriguing question of what biochemical process in the nucleus makes all nervous system-associated cells different from cells outside the nervous system.

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