Development of Tissue- and Time-specific Gene Knockout in Mice

2007 ◽  
Vol 13 (6) ◽  
pp. S10
Author(s):  
Satoshi Sakai
Keyword(s):  
Gene Knockout ◽  
Specific Gene ◽  
Time Specific

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 Rapid generation of conditional knockout mice using the CRISPR-Cas9 system and electroporation for neuroscience research

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Hirofumi Nishizono ◽  
Yuki Hayano ◽  
Yoshihisa Nakahata ◽  
Yasuhito Ishigaki ◽  
Ryohei Yasuda
Keyword(s):  
Function Analysis ◽  
Low Cost ◽  
Conditional Knockout ◽  
Specific Gene ◽  
Time Saving ◽  
Vitro Fertilization ◽  
Short Period ◽  
Genome Analyses ◽  
Time Specific

AbstractThe Cre/LoxP-based conditional knockout technology is a powerful tool for gene function analysis that allows region- and time-specific gene manipulation. However, inserting a pair of LoxP cassettes to generate conditional knockout can be technically challenging and thus time- and resource-consuming. This study proposes an efficient, low-cost method to generate floxed mice using in vitro fertilization and the CRISPR-Cas9 system over two consecutive generations. This method allowed us to produce floxed mice targeting exons 5 and 6 of CaMK1 in a short period of 125 days, using only 16 mice. In addition, we directly edited the genome of fertilized eggs of mice with our target genetic background, C57BL/6 N, to eliminate additional backcrossing steps. We confirmed that the genome of the generated floxed mice was responsive to the Cre protein. This low-cost, time-saving method for generating conditional knockout will facilitate comprehensive, tissue-specific genome analyses.

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 Diurnal.plant.tools: comparative transcriptomic and co-expression analyses of diurnal gene expression of the Archaeplastida kingdom

2019 ◽  
Author(s):  
Jonathan Wei Xiong Ng ◽  
Qiao Wen Tan ◽  
Camilla Ferrari ◽  
Marek Mutwil
Keyword(s):  
Gene Expression ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Specific Gene ◽  
Species Comparisons ◽  
Gene Modules ◽  
Time Specific ◽  
Almost All ◽  
User Friendly ◽  
Diurnal Regulation

ABSTRACTAlmost all organisms coordinate some aspects of their biology through the diurnal cycle. Photosynthetic organisms, and plants especially, have established complex programs that coordinate physiological, metabolic and developmental processes with the changing light. The diurnal regulation of the underlying transcriptional processes is observed when groups of functionally related genes (gene modules) are expressed at a specific time of the day. However, studying the diurnal regulation of these gene modules in the plant kingdom was hampered by the large amount of data required for the analyses. To meet this need, we used gene expression data from 17 diurnal studies spanning the whole Archaeplastida kingdom (Plantae kingdom in the broad sense) to make an online diurnal database. We have equipped the database with tools that allow user-friendly cross-species comparisons of gene expression profiles, entire co-expression networks, co-expressed clusters (involved in specific biological processes), time-specific gene expression, and others. We exemplify how these tools can be used by studying three important biological questions: (i) the evolution of cell division, (ii) the diurnal control of gene modules in algae and (iii) the conservation of diurnally-controlled modules across species. The database is freely available at http://diurnal.plant.tools/.

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.

scholarly journals Systems-level analysis identifies key regulators driving epileptogenesis in temporal lobe epilepsy

2019 ◽  
Author(s):  
Yingxue Fu ◽  
Zihu Guo ◽  
Ziyin Wu ◽  
Liyang Chen ◽  
Yaohua Ma ◽  
...  
Keyword(s):  
Temporal Lobe Epilepsy ◽  
Temporal Lobe ◽  
Hippocampal Sclerosis ◽  
Coexpression Network ◽  
Specific Gene ◽  
Protein Activity ◽  
New Gene ◽  
Cell Type Specific ◽  
Time Specific ◽  
Level Analysis

AbstractTemporal lobe epilepsy (TLE) is the most prevalent and often devastating form of epilepsy. The molecular mechanism underlying the development of TLE remains largely unknown, which hinders the discovery of effective anti-epileptogenic drugs. In this study, we built a systems-level analytic framework which integrates gene meta-signatures, gene coexpression network and cellular regulatory network to unveil the evolution landscape of epileptogenic process and to identify key regulators that govern the transition between different epileptogenesis stages. The time-specific hippocampal transcriptomic profiles from five independent rodent TLE models were grouped into acute, latent and chronic stages of epileptogenesis, and were utilized for generating stage-specific gene expression signatures. 13 cell-type specific functional modules were identified from the epilepsy-context coexpression network, and five of them were significantly associated with the entire epileptogenic process. By inferring the differential protein activity of gene regulators in each stage, 265 key regulators underlying epileptogenesis were obtained. Among them, 122 regulators were demonstrated being associated with high seizure frequency and/or hippocampal sclerosis in human TLE patients. Importantly, we discovered four new gene regulators (ANXA5, FAM107A, SEPT2 and SPARC) whose upregulation may drive the process of epileptogenesis and further lead to chronic recurrent seizures or hippocampal sclerosis. Our findings provide a landscape of the gene network dynamics underlying epileptogenesis and uncovered candidate regulators that may serve as potential targets for future anti-epileptogenic therapy development.

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.

Sign in / Sign up

Export Citation Format

Share Document