scholarly journals AAV-Mediated CRISPRi and RNAi Based Gene Silencing in Mouse Hippocampal Neurons

Cells ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 324
Author(s):  
Matthias Deutsch ◽  
Anne Günther ◽  
Rodrigo Lerchundi ◽  
Christine R. Rose ◽  
Sabine Balfanz ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Initiation ◽  
Hippocampal Neurons ◽  
De Novo ◽  
Protein Biosynthesis ◽  
Physiological Role ◽  
High Specificity ◽  
Hcn Channels ◽  
Proliferating Cells ◽  
Neuronal Tissues

Uncovering the physiological role of individual proteins that are part of the intricate process of cellular signaling is often a complex and challenging task. A straightforward strategy of studying a protein’s function is by manipulating the expression rate of its gene. In recent years, the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9-based technology was established as a powerful gene-editing tool for generating sequence specific changes in proliferating cells. However, obtaining homogeneous populations of transgenic post-mitotic neurons by CRISPR/Cas9 turned out to be challenging. These constraints can be partially overcome by CRISPR interference (CRISPRi), which mediates the inhibition of gene expression by competing with the transcription machinery for promoter binding and, thus, transcription initiation. Notably, CRISPR/Cas is only one of several described approaches for the manipulation of gene expression. Here, we targeted neurons with recombinant Adeno-associated viruses to induce either CRISPRi or RNA interference (RNAi), a well-established method for impairing de novo protein biosynthesis by using cellular regulatory mechanisms that induce the degradation of pre-existing mRNA. We specifically targeted hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels, which are widely expressed in neuronal tissues and play essential physiological roles in maintaining biophysical characteristics in neurons. Both of the strategies reduced the expression levels of three HCN isoforms (HCN1, 2, and 4) with high specificity. Furthermore, detailed analysis revealed that the knock-down of just a single HCN isoform (HCN4) in hippocampal neurons did not affect basic electrical parameters of transduced neurons, whereas substantial changes emerged in HCN-current specific properties.

scholarly journals Transcriptional signatures of somatic neoblasts and germline cells in Macrostomum lignano

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Magda Grudniewska ◽  
Stijn Mouton ◽  
Daniil Simanov ◽  
Frank Beltman ◽  
Margriet Grelling ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
De Novo ◽  
Transcriptome Assembly ◽  
Model Organism ◽  
Proliferating Cells ◽  
Macrostomum Lignano ◽  
Germline Cells

The regeneration-capable flatworm Macrostomum lignano is a powerful model organism to study the biology of stem cells in vivo. As a flatworm amenable to transgenesis, it complements the historically used planarian flatworm models, such as Schmidtea mediterranea. However, information on the transcriptome and markers of stem cells in M. lignano is limited. We generated a de novo transcriptome assembly and performed the first comprehensive characterization of gene expression in the proliferating cells of M. lignano, represented by somatic stem cells, called neoblasts, and germline cells. Knockdown of a selected set of neoblast genes, including Mlig-ddx39, Mlig-rrm1, Mlig-rpa3, Mlig-cdk1, and Mlig-h2a, confirmed their crucial role for the functionality of somatic neoblasts during homeostasis and regeneration. The generated M. lignano transcriptome assembly and gene expression signatures of somatic neoblasts and germline cells will be a valuable resource for future molecular studies in M. lignano.

scholarly journals Promoter architecture and sex-specific gene expression in the microcrustacean Daphnia pulex revealed by large-scale profiling of 5′-mRNA ends

2016 ◽  
Author(s):  
R. Taylor Raborn ◽  
Ken Spitze ◽  
Volker P. Brendel ◽  
Michael Lynch
Keyword(s):  
Gene Expression ◽  
Transcription Initiation ◽  
Large Scale ◽  
De Novo ◽  
Core Promoter ◽  
Daphnia Pulex ◽  
Model Systems ◽  
Specific Gene ◽  
Promoter Architecture ◽  
A Genome

AbstractLarge-scale TSS profiling produces a high-resolution, quantitative picture of transcription initiation and core promoter locations within a genome. However, application of TSS profiling to date has largely been restricted to a small set of prominent model systems. We sought to characterize the cis-regulatory landscape of the water flea Daphnia pulex, an emerging model arthropod that reproduces both asexually (via parthenogenesis) and sexually (via meiosis). We performed CAGE with RNA isolated from D. pulex within three developmental states: sexual females, asexual females, and males. Identified TSSs were utilized to generate a ‘Daphnia Promoter Atlas’-a catalog of active promoters across the surveyed states. We carried out de novo motif discovery using CAGE-defined TSSs and identified eight candidate core promoter motifs; this collection includes canonical promoter elements (e.g. TATA, Initiator) in addition to others lacking obvious orthologs. A comparison of promoter activities found evidence for considerable state-specific differential gene expression between states. Our work represents the first global definition of transcription initiation and promoter architecture in crustaceans. The Daphnia Promoter Atlas presented here provides a valuable resource for comparative study of cis-regulatory regions in arthropods, as well as for investigations into the circuitries that underpin meiosis and parthenogenesis.

scholarly journals Human Immunodeficiency Virus Type 1 (HIV-1) Vpr Functions as an Immediate-Early Protein during HIV-1 Infection

1999 ◽  
Vol 73 (5) ◽  
pp. 4101-4109 ◽  
Author(s):  
Mohammed Hrimech ◽  
Xiao-Jian Yao ◽  
François Bachand ◽  
Nicole Rougeau ◽  
Éric A. Cohen
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Human Immunodeficiency Virus ◽  
De Novo ◽  
Proliferating Cells ◽  
Early Protein ◽  
Immunodeficiency Virus ◽  
A Cell ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) Vpr is a virion-associated protein which facilitates HIV-1 infection of nondividing cells by contributing to the nuclear transport of the preintegration complex (PIC). Vpr was also shown to induce a cell cycle G2 arrest in infected proliferating cells that optimizes HIV-1 long terminal repeat (LTR)-directed gene expression and viral production. However, it is unclear whether this activity is mediated primarily early by virion-associated Vpr or alternatively late during infection when Vpr is de novo expressed. We report here that in the absence of de novo expression, virion-associated Vpr induces a transient G2 arrest that can subsequently lead to cell killing by apoptosis. Interestingly, the induction of both cell cycle G2 arrest and apoptosis by virion-associated Vpr requires viral entry but not viral replication, since reverse transcriptase and protease inhibitor treatments do not prevent these Vpr effects. These results raise the possibility that in vivo both infectious and noninfectious viruses contribute to the dysfunction and killing of CD4+ cells. In addition, our results reveal that virion-associated Vpr stimulates viral replication in proliferating cells after establishing a cell cycle G2 arrest by increasing LTR-directed gene expression. Importantly, this Vpr-mediated LTR activation appears to be a requirement for subsequent optimal Tat transactivation. Taken together, these results strongly suggest that in addition to participating in the HIV PIC nuclear transport in nondividing cells, virion-associated Vpr activates HIV-1 LTR-directed gene expression by manipulating the host cell cycle. From this, we conclude that Vpr functions as an immediate-early protein during HIV-1 infection.

Untangling the Metabolic Reprogramming in Brain Cancer: Discovering Key Molecular Players Using Mass Spectrometry

2019 ◽  
Vol 19 (17) ◽  
pp. 1521-1534 ◽  
Author(s):  
Anatoly Sorokin ◽  
Vsevolod Shurkhay ◽  
Stanislav Pekov ◽  
Evgeny Zhvansky ◽  
Daniil Ivanov ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Brain Cancer ◽  
De Novo ◽  
Aerobic Glycolysis ◽  
Lipid Production ◽  
Dietary Fatty Acids ◽  
Metabolic Reprogramming ◽  
Detection Methods ◽  
Malignant Cells ◽  
Proliferating Cells

Cells metabolism alteration is the new hallmark of cancer, as well as an important method for carcinogenesis investigation. It is well known that the malignant cells switch to aerobic glycolysis pathway occurring also in healthy proliferating cells. Recently, it was shown that in malignant cells de novo synthesis of the intracellular fatty acid replaces dietary fatty acids which change the lipid composition of cancer cells noticeably. These alterations in energy metabolism and structural lipid production explain the high proliferation rate of malignant tissues. However, metabolic reprogramming affects not only lipid metabolism but many of the metabolic pathways in the cell. 2-hydroxyglutarate was considered as cancer cell biomarker and its presence is associated with oxidative stress influencing the mitochondria functions. Among the variety of metabolite detection methods, mass spectrometry stands out as the most effective method for simultaneous identification and quantification of the metabolites. As the metabolic reprogramming is tightly connected with epigenetics and signaling modifications, the evaluation of metabolite alterations in cells is a promising approach to investigate the carcinogenesis which is necessary for improving current diagnostic capabilities and therapeutic capabilities. In this paper, we overview recent studies on metabolic alteration and oncometabolites, especially concerning brain cancer and mass spectrometry approaches which are now in use for the investigation of the metabolic pathway.

scholarly journals Malpigmentation of Common Sole (Solea solea) during Metamorphosis Is Associated with Differential Synaptic-Related Gene Expression

Animals ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 2273
Author(s):  
Menelaos Kavouras ◽  
Emmanouil E. Malandrakis ◽  
Ewout Blom ◽  
Kyriaki Tsilika ◽  
Theodoros Danis ◽  
...  
Keyword(s):  
Gene Expression ◽  
Nervous System ◽  
De Novo ◽  
Close Association ◽  
Ionic Channels ◽  
Long Term Potentiation ◽  
General Term ◽  
The Difference ◽  
Common Sole

In farmed flatfish, such as common sole, color disturbances are common. Dyschromia is a general term that includes the color defects on the blind and ocular sides of the fish. The purpose was to examine the difference in gene expression between normal pigmented and juveniles who present ambicoloration. The analysis was carried out with next-generation sequencing techniques and de novo assembly of the transcriptome. Transcripts that showed significant differences (FDR < 0.05) in the expression between the two groups, were related to those of zebrafish (Danio rerio), functionally identified, and classified into categories of the gene ontology. The results revealed that ambicolorated juveniles exhibit a divergent function, mainly of the central nervous system at the synaptic level, as well as the ionic channels. The close association of chromophore cells with the growth of nerve cells and the nervous system was recorded. The pathway, glutamate binding–activation of AMPA and NMDA receptors–long-term stimulation of postsynaptic potential–LTP (long term potentiation)–plasticity of synapses, appears to be affected. In addition, the development of synapses also seems to be affected by the interaction of the LGI (leucine-rich glioma inactivated) protein family with the ADAM (a disintegrin and metalloprotease) ones.

scholarly journals The brain transcriptome of the wolf spider, Schizocosa ocreata

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Daniel Stribling ◽  
Peter L. Chang ◽  
Justin E. Dalton ◽  
Christopher A. Conow ◽  
Malcolm Rosenthal ◽  
...  
Keyword(s):  
Gene Expression ◽  
De Novo ◽  
Transcriptome Assembly ◽  
Model Organisms ◽  
De Novo Transcriptome Assembly ◽  
De Novo Transcriptome ◽  
Wolf Spiders ◽  
Schizocosa Ocreata ◽  
Genomic Studies ◽  
The Brain

Abstract Objectives Arachnids have fascinating and unique biology, particularly for questions on sex differences and behavior, creating the potential for development of powerful emerging models in this group. Recent advances in genomic techniques have paved the way for a significant increase in the breadth of genomic studies in non-model organisms. One growing area of research is comparative transcriptomics. When phylogenetic relationships to model organisms are known, comparative genomic studies provide context for analysis of homologous genes and pathways. The goal of this study was to lay the groundwork for comparative transcriptomics of sex differences in the brain of wolf spiders, a non-model organism of the pyhlum Euarthropoda, by generating transcriptomes and analyzing gene expression. Data description To examine sex-differential gene expression, short read transcript sequencing and de novo transcriptome assembly were performed. Messenger RNA was isolated from brain tissue of male and female subadult and mature wolf spiders (Schizocosa ocreata). The raw data consist of sequences for the two different life stages in each sex. Computational analyses on these data include de novo transcriptome assembly and differential expression analyses. Sample-specific and combined transcriptomes, gene annotations, and differential expression results are described in this data note and are available from publicly-available databases.

scholarly journals Transcription initiation as a control point in plastid gene expression

2021 ◽  
Vol 1864 (3) ◽  
pp. 194689
Author(s):  
Sujith Puthiyaveetil ◽  
Steven D. McKenzie ◽  
Gilbert E. Kayanja ◽  
Iskander M. Ibrahim
Keyword(s):  
Gene Expression ◽  
Transcription Initiation ◽  
Control Point ◽  
Plastid Gene ◽  
Plastid Gene Expression

Serial analysis of gene expression (SAGE) during porcine embryo development

2004 ◽  
Vol 16 (2) ◽  
pp. 87 ◽  
Author(s):  
Le Ann Blomberg ◽  
Kurt A. Zuelke
Keyword(s):  
Gene Expression ◽  
Functional Genomics ◽  
Embryo Development ◽  
Molecular Mechanisms ◽  
Physiological Role ◽  
Transgenic Animals ◽  
Specific Gene ◽  
Research Strategies

Functional genomics provides a powerful means for delving into the molecular mechanisms involved in pre-implantation development of porcine embryos. High rates of embryonic mortality (30%), following either natural mating or artificial insemination, emphasise the need to improve the efficiency of reproduction in the pig. The poor success rate of live offspring from in vitro-manipulated pig embryos also hampers efforts to generate transgenic animals for biotechnology applications. Previous analysis of differential gene expression has demonstrated stage-specific gene expression for in vivo-derived embryos and altered gene expression for in vitro-derived embryos. However, the methods used to date examine relatively few genes simultaneously and, thus, provide an incomplete glimpse of the physiological role of these genes during embryogenesis. The present review will focus on two aspects of applying functional genomics research strategies for analysing the expression of genes during elongation of pig embryos between gestational day (D) 11 and D12. First, we compare and contrast current methodologies that are being used for gene discovery and expression analysis during pig embryo development. Second, we establish a paradigm for applying serial analysis of gene expression as a functional genomics tool to obtain preliminary information essential for discovering the physiological mechanisms by which distinct embryonic phenotypes are derived.

scholarly journals Viral Gene Expression Patterns in Human Herpesvirus 6B-Infected T Cells

2002 ◽  
Vol 76 (15) ◽  
pp. 7578-7586 ◽  
Author(s):  
Bodil Øster ◽  
Per Höllsberg
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Protein Synthesis ◽  
De Novo ◽  
Expression Patterns ◽  
Human Herpesvirus ◽  
Viral Gene Expression ◽  
Polymerase Activity ◽  
Viral Gene ◽  
De Novo Protein Synthesis

ABSTRACT Herpesvirus gene expression is divided into immediate-early (IE) or α genes, early (E) or β genes, and late (L) or γ genes on the basis of temporal expression and dependency on other gene products. By using real-time PCR, we have investigated the expression of 35 human herpesvirus 6B (HHV-6B) genes in T cells infected by strain PL-1. Kinetic analysis and dependency on de novo protein synthesis and viral DNA polymerase activity suggest that the HHV-6B genes segregate into six separate kinetic groups. The genes expressed early (groups I and II) and late (groups V and VI) corresponded well with IE and L genes, whereas the intermediate groups III and IV contained E and L genes. Although HHV-6B has characteristics similar to those of other roseoloviruses in its overall gene regulation, we detected three B-variant-specific IE genes. Moreover, genes that were independent of de novo protein synthesis clustered in an area of the viral genome that has the lowest identity to the HHV-6A variant. The organization of IE genes in an area of the genome that differs from that of HHV-6A underscores the distinct differences between HHV-6B and HHV-6A and may provide a basis for further molecular and immunological analyses to elucidate their different biological behaviors.

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