Effect of a specific-gene knockout on metabolism

2013 ◽  
pp. 359-447
Author(s):  
Kazuyuki Shimizu
Keyword(s):  
Gene Knockout ◽  
Specific Gene

Abstract 102: Time-of-intake Regulates Glucocorticoid Pharmacology Of Cardiac Bioenergetics

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Mattia Quattrocelli ◽  
Michelle Wintzinger ◽  
Karen Miz
Keyword(s):  
Ex Vivo ◽  
Gene Knockout ◽  
Heart Tissue ◽  
Functional Coupling ◽  
Specific Gene ◽  
Heart Metabolism ◽  
Respiratory Capacity ◽  
Circadian Time ◽  
The Impact ◽  
Mitochondrial Respiratory

Glucocorticoid steroids are circadian regulators of energy balance. However, the specific direct effects of glucocorticoids on heart metabolism remain unresolved. Moreover, the impact of circadian time-of-intake on glucocorticoid pharmacology is still unknown. Here, we investigated whether circadian time of exposure gates the effects of synthetic glucocorticoids on heart bioenergetics. We compared the effects of diurnal versus nocturnal glucocorticoids in heart tissue and mitochondria from wildtype mice, controlling the subjective circadian time of drug injection. To avoid interferences from other tissues, we developed an ex vivo system to interrogate the mitochondrial respiratory capacity rate (state III/state IV) in isolated hearts. We found that diurnal but not nocturnal pulse of the glucocorticoid prednisone increased the mitochondrial respiratory capacity rate in heart. This correlated with circadian-restricted effects on mitochondrial abundance. This was remarkable as it contrasts the circadian fluctuations of endogenous glucocorticoids. Using transgenic mice with inducible cardiac-specific gene knockout, we found that the bioenergetic effects of diurnal-restricted prednisone were dependent on the glucocorticoid receptor and its co-factor Kruppel-like factor 15. Considering the bioenergetic decline that hallmarks the aging heart, we asked whether these circadian-gated effects were applicable to aged mice. We therefore treated 24 months-old mice for 12 weeks with a diurnal-restricted regimen of prednisone. Compared to vehicle, diurnal prednisone increased mitochondrial respiration along with NAD + and ATP content in aged hearts. Moreover, lipidomic profiling of myocardial tissue showed that the vast majority of lipids were downregulated after treatment, including triacylglycerols, suggesting a functional coupling between lipid utilization and mitochondrial oxidation in treated hearts. We also found that diurnal-restricted prednisone rescued bioenergetics and improved function in diabetic hearts from db/db mice. In summary, our data indicate that glucocorticoids regulate cardiac bioenergetics according to circadian-time of intake, supporting a role for chrono-pharmacology in aged and diabetic hearts.

scholarly journals Improved Experimental Procedures for Achieving Efficient Germ Line Transmission of Nonobese Diabetic (NOD)-Derived Embryonic Stem Cells

2004 ◽  
Vol 5 (3) ◽  
pp. 219-226 ◽  
Author(s):  
Satoko Arai ◽  
Christina Minjares ◽  
Seiho Nagafuchi ◽  
Toru Miyazaki
Keyword(s):  
High Efficiency ◽  
Germ Line ◽  
Gene Knockout ◽  
Es Cells ◽  
Embryonic Stem ◽  
Nod Mice ◽  
Insulin Dependent Diabetes Mellitus ◽  
Transmission Efficiency ◽  
Dependent Diabetes Mellitus ◽  
Specific Gene

The manipulation of a specific gene in NOD mice, the best animal model for insulin-dependent diabetes mellitus (IDDM), must allow for the precise characterization of the functional involvement of its encoded molecule in the pathogenesis of the disease. Although this has been attempted by the cross-breeding of NOD mice with many gene knockout mice originally created on the 129 or C57BL/6 strain background, the interpretation of the resulting phenotype(s) has often been confusing due to the possibility of a known or unknown disease susceptibility locus (e.g.,Iddlocus) cosegregating with the targeted gene from the diabetes-resistant strain. Therefore, it is important to generate mutant mice on a pure NOD background by using NOD-derived embryonic stem (ES) cells. By using the NOD ES cell line established by Nagafuchi and colleagues in 1999 (FEBSLett., 455, 101–104), the authors reexamined various conditions in the context of cell culture, DNA transfection, and blastocyst injection, and achieved a markedly improved transmission efficiency of these NOD ES cells into the mouse germ line. These modifications will enable gene targeting on a “pure” NOD background with high efficiency, and contribute to clarifying the physiological roles of a variety of genes in the disease course of IDDM.

scholarly journals Ursolic Acid Inhibits the Activation of Kupffer Cells by Caspase-11/NLRP3 Inflammasome Signaling Pathways

2021 ◽  
Author(s):  
Yuan Nie ◽  
Chen-kai Huang ◽  
Cong Liu ◽  
Xuan Zhu
Keyword(s):  
Liver Fibrosis ◽  
Ursolic Acid ◽  
Liver Damage ◽  
Kupffer Cells ◽  
Knockout Mice ◽  
Nlrp3 Inflammasome ◽  
Gene Knockout ◽  
Specific Gene

Abstract Background: Previous studies have indicated that Kupffer cells (KCs) are the main regulatory cells for the activation of hepatic stellate cells (HSCs), and caspase-11/NLRP3 inflammasome signaling plays crucial roles in the activation of monocyte-macrophages. Ursolic acid (UA) is a traditional Chinese medicine with antifibrotic effects, but the molecular mechanism underlying these effects is still unclear.Methods: A mouse primary Kupffer cell line in vitro and liver fibrosis mice (including specific gene knockout mice) in vivo were selected as experimental objects. RT-qPCR and Western blotting techniques were utilized to assess the mRNA and protein expression in each group. ELISA and histological analysis were utilized to assess liver injury and collagen deposition.Results: In vitro, caspase-11/NLRP3 inflammasome signaling promoted the activation of Kupffer cells, and UA inhibited the activation of Kupffer cells by caspase-11/NLRP3 inflammasome signaling. In vivo, UA reversed liver damage and fibrosis in fibrotic mice and was related to Kupffer cells; the expression of Caspase-11/NLRP3 inflammasome signaling in Kupffer cells of the UA group was inhibited. Even in the CCl4 group, the liver damage and fibrosis of NLRP3 knockout mice were alleviated, and related experiments also proved that the inhibitory effect of UA on Kupffer cells was related to the activation of the NLRP3 inflammasome.Conclusion: Caspase-11/NLRP3 inflammasome signal transduction is closely related to the activation of Kupffer cells and the occurrence of liver fibrosis. Additionally, caspase-11/NLRP3 inflammasome signaling serves as a new target for UA antifibrosis treatment.

scholarly journals Extension of Endocardium-Derived Vessels Generate Coronary Arteries in Neonates

2022 ◽  
Author(s):  
Juan Tang ◽  
Huan Zhu ◽  
Xueying Tian ◽  
Haixiao Wang ◽  
Shaoyan Liu ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Coronary Artery ◽  
Coronary Arteries ◽  
Molecular Mechanisms ◽  
Gene Knockout ◽  
Lineage Tracing ◽  
Specific Gene ◽  
Genetic Lineage ◽  
Light Sheet Microscopy ◽  
Neonatal Heart

Background: Unraveling how new coronary arteries develop may provide critical information for establishing novel therapeutic approaches to treating ischemic cardiac diseases. There are two distinct coronary vascular populations derived from different origins in the developing heart. Understanding the formation of coronary arteries may provide insights into new ways of promoting coronary artery formation after myocardial infarction. Methods: To understand how intramyocardial coronary arteries are generated to connect these two coronary vascular populations, we combined genetic lineage tracing, light-sheet microscopy, fluorescence micro-optical sectioning tomography, and tissue-specific gene knockout approaches to understand their cellular and molecular mechanisms. Results: We show that a subset of intramyocardial coronary arteries form by angiogenic extension of endocardium-derived vascular tunnels in the neonatal heart. Three-dimensional whole-mount fluorescence imaging showed that these endocardium-derived vascular tunnels or tubes adopt an arterial fate in neonates. Mechanistically, we implicate Mettl3 and Notch signaling in regulating endocardium-derived intramyocardial coronary artery formation. Functionally, these intramyocardial arteries persist into adulthood and play a protective role after myocardial infarction. Conclusions: A subset of intramyocardial coronary arteries form by extension of endocardium-derived vascular tunnels in the neonatal heart.

Isoform switching from SM-B to SM-A myosin results in decreased contractility and altered expression of thin filament regulatory proteins

2004 ◽  
Vol 287 (3) ◽  
pp. C723-C729 ◽  
Author(s):  
Gopal J. Babu ◽  
Gail J. Pyne ◽  
Yingbi Zhou ◽  
Chris Okwuchukuasanya ◽  
Joseph E. Brayden ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Thin Filament ◽  
Isometric Force ◽  
Gene Knockout ◽  
Regulatory Proteins ◽  
Mitogen Activated Protein Kinase ◽  
Specific Gene ◽  
Critical Determinant ◽  
Altered Expression ◽  
Mesenteric Vessels

We previously generated an isoform-specific gene knockout mouse in which SM-B myosin is permanently replaced by SM-A myosin. In this study, we examined the effects of SM-B myosin loss on the contractile properties of vascular smooth muscle, specifically peripheral mesenteric vessels and aorta. The absence of SM-B myosin leads to decreased velocity of shortening and increased isometric force generation in mesenteric vessels. Surprisingly, the same changes occur in aorta, which contains little or no SM-B myosin in wild-type animals. Calponin and activated mitogen-activated protein kinase expression is increased and caldesmon expression is decreased in aorta, as well as in bladder. Light chain-17b isoform (LC17b) expression is increased in aorta. These results suggest that the presence or absence of SM-B myosin is a critical determinant of smooth muscle contraction and that its loss leads to additional changes in thin filament regulatory proteins.

scholarly journals The non-redundant nature of the Axin2 regulatory network in the canonical Wnt signaling pathway

2021 ◽  
Author(s):  
Ana R Moshkovsky ◽  
Marc W Kirschner
Keyword(s):  
Wnt Pathway ◽  
Gene Knockout ◽  
Gene Activation ◽  
Wnt Signaling Pathway ◽  
Differential Regulation ◽  
Specific Gene ◽  
Redundant Gene ◽  
Specific Regulation ◽  
Differential Responsiveness ◽  
Canonical Wnt

Axin is one of two essential scaffolds in the canonical Wnt pathway that converts signals at the plasma membrane to signals inhibiting the degradation of β-catenin, leading to its accumulation and specific gene activation. In vertebrates there are two forms of Axin, Axin1 and Axin2, which are similar at the protein level and genetically redundant. We show here that differential regulation of the two genes on the transcriptional and proteostatic level confers robustness and differential responsiveness that can be used in tissue specific regulation. Such subtle features may distinguish other redundant gene pairs that are commonly found in vertebrates through gene knockout experiments.

scholarly journals Expanded FLP toolbox for spatiotemporal protein degradation and transcriptomic profiling in C. elegans

2021 ◽  
Author(s):  
Adrian Fragoso-Luna ◽  
Cristina Ayuso ◽  
Michael Eibl ◽  
Celia Munoz-Jimenez ◽  
Vladimir Benes ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Degradation ◽  
Gene Function ◽  
Gene Knockout ◽  
Added Value ◽  
Nuclear Pore ◽  
Specific Gene ◽  
Transcriptomic Profiling ◽  
Control Of Gene Expression ◽  
C Elegans

Control of gene expression in specific tissues and/or at certain stages of development allows the study and manipulation of gene function with high precision. Site-specific genome recombination by the Flippase (FLP) and Cre enzymes has proven particularly relevant. Joint efforts of many research groups have led to the creation of efficient FLP and Cre drivers to regulate gene expression in a variety of tissues in Caenorhabditis elegans. Here, we extend this toolkit by the addition of FLP lines that drive recombination specifically in distal tip cells, the somatic gonad, coelomocytes and the epithelial P lineage. In some cases, recombination-mediated gene knockouts do not completely deplete protein levels due to persistence of long-lived proteins. To overcome this, we developed a spatiotemporally regulated degradation system for GFP fusion proteins (GFPdeg) based on FLP-mediated recombination. Using two stable nuclear pore proteins, MEL-28/ELYS and NPP-2/NUP85 as examples, we report the benefit of combining tissue-specific gene knockout and protein degradation to achieve complete protein depletion. We also demonstrate that FLP-mediated recombination can be utilized to identify nascent transcripts in a tissue of interest. We have adapted thiol(SH)-linked alkylation for the metabolic sequencing of RNA in tissue (SLAM-ITseq) for C. elegans. By focusing on a well-characterized tissue, the hypodermis, we show that the vast majority of genes identified by SLAM-ITseq are known to be expressed in this tissue, but with the added value of temporal resolution. These tools allow combining FLP activity for simultaneous gene inactivation and transcriptomic profiling, thus enabling the inquiry of gene function in various complex biological processes.

scholarly journals CRISPR-TRAPSeq identifies the QKI RNA binding protein as important for astrocytic maturation and control of thalamocortical synapses

2020 ◽  
Author(s):  
Kristina Sakers ◽  
Yating Liu ◽  
Lorida Llaci ◽  
Michael J. Vasek ◽  
Michael A. Rieger ◽  
...  
Keyword(s):  
Rna Binding ◽  
Gene Knockout ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Cell Types ◽  
Specific Gene ◽  
Specific Cell ◽  
Normal Brain ◽  
Oligodendrocyte Development

AbstractQuaking RNA binding protein(QKI) is essential for oligodendrocyte development as myelination requires MBP mRNA regulation and localization by the cytoplasmic isoforms(e.g. QKI-6). QKI-6 is also highly expressed in astrocytes, which were recently demonstrated to have regulated mRNA localization. Here, we show via CLIPseq that QKI-6 binds 3’ UTRs of a subset of astrocytic mRNAs, including many enriched in peripheral processes. Binding is enriched near stop codons, which is mediated partially by QKI binding motifs(QBMs) yet spreads to adjacent sequences. We developed CRISPR TRAPseq: a viral approach for mosaic, cell-type specific gene mutation with simultaneous translational profiling. This enabled study of QKI-deleted astrocytes in an otherwise normal brain. Astrocyte-targeted QKI deletion altered translation and maturation, while also increasing synaptic density within the astrocyte’s territory. Overall, our data indicate QKI is required for astrocyte maturation and demonstrate an approach for a highly targeted translational assessment of gene knockout in specific cell-types in vivo.

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