scholarly journals The CRE luc Bioluminescence Transgenic Mouse Model for Detecting Ligand Activation of GPCRs

2013 ◽  
Vol 19 (2) ◽  
pp. 232-241 ◽  
Author(s):  
Holly Dressler ◽  
Kyriakos Economides ◽  
Sarah Favara ◽  
Nancy N. Wu ◽  
Zhen Pang ◽  
...  
Keyword(s):  
Mouse Model ◽  
Transgenic Mouse ◽  
Expression Profiles ◽  
Tissue Expression ◽  
Transgenic Mouse Model ◽  
Luciferase Reporter ◽  
Primary Cells ◽  
Compound Libraries ◽  
Cellular Environment

Numerous assays have been developed to investigate the interactions between G-protein–coupled receptors (GPCRs) and their ligands since GPCRs are key therapeutic targets. Reporter-based assays using the cAMP response element (CRE) coupled with bioluminescence from a luciferase reporter have been used extensively in vitro with high-throughput screens (HTS) of large chemical compound libraries. We have generated a transgenic mouse model (CRE luc) with a luciferase reporter under the control of a synthetic promoter that contains several CREs, which supports real-time bioimaging of GPCR ligand activity in whole animals, tissues, or primary cells. In the CRE luc model, GPCR signaling through the cAMP pathway can be detected from the target GPCR that is in a native cellular environment with a full complement of associated receptors and membrane constituents. Multiple independent lines have been produced by random integration of the transgene, resulting in tissue expression profiles covering the major organs. The goal of the CRE luc model is to accelerate the transition from HTS to profiling of GPCR small-molecule leads in preclinical animal disease models, as well as define the mechanism of action of GPCR drugs in three experimental formats: primary cells, tissue homogenates, and whole animals.

scholarly journals A Transgenic Mouse Model for High Content, Cell Cycle Phenotype Screening in Live Primary Cells

Cell Cycle ◽  
2007 ◽  
Vol 6 (18) ◽  
pp. 2276-2283 ◽  
Author(s):  
Richard O. Burney ◽  
Alan I. Lee ◽  
Denise E. Leong ◽  
Joshua T. Jones ◽  
Angela T. Hahn ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Mouse Model ◽  
Transgenic Mouse ◽  
Transgenic Mouse Model ◽  
Primary Cells ◽  
Phenotype Screening

scholarly journals Conserved Asp-137 Is Important for both Structure and Regulatory Functions of Cardiac α-Tropomyosin (α-TM) in a Novel Transgenic Mouse Model Expressing α-TM-D137L

2013 ◽  
Vol 288 (23) ◽  
pp. 16235-16246 ◽  
Author(s):  
Sumeyye Yar ◽  
Shamim A. K. Chowdhury ◽  
Robert T. Davis ◽  
Minae Kobayashi ◽  
Michelle M. Monasky ◽  
...  
Keyword(s):  
Mouse Model ◽  
Transgenic Mouse ◽  
Coiled Coil ◽  
Transgenic Mouse Model ◽  
Hydrophobic Core ◽  
Scanning Calorimetry ◽  
Physiological Importance ◽  
Cardiomyocyte Contractility ◽  
Regulatory Functions

α-Tropomyosin (α-TM) has a conserved, charged Asp-137 residue located in the hydrophobic core of its coiled-coil structure, which is unusual in that the residue is found at a position typically occupied by a hydrophobic residue. Asp-137 is thought to destabilize the coiled-coil and so impart structural flexibility to the molecule, which is believed to be crucial for its function in the heart. A previous in vitro study indicated that the conversion of Asp-137 to a more typical canonical Leu alters flexibility of TM and affects its in vitro regulatory functions. However, the physiological importance of the residue Asp-137 and altered TM flexibility is unknown. In this study, we further analyzed structural properties of the α-TM-D137L variant and addressed the physiological importance of TM flexibility in cardiac function in studies with a novel transgenic mouse model expressing α-TM-D137L in the heart. Our NMR spectroscopy data indicated that the presence of D137L introduced long range rearrangements in TM structure. Differential scanning calorimetry measurements demonstrated that α-TM-D137L has higher thermal stability compared with α-TM, which correlated with decreased flexibility. Hearts of transgenic mice expressing α-TM-D137L showed systolic and diastolic dysfunction with decreased myofilament Ca2+ sensitivity and cardiomyocyte contractility without changes in intracellular Ca2+ transients or post-translational modifications of major myofilament proteins. We conclude that conversion of the highly conserved Asp-137 to Leu results in loss of flexibility of TM that is important for its regulatory functions in mouse hearts. Thus, our results provide insight into the link between flexibility of TM and its function in ejecting hearts.

scholarly journals Optogenetic Modulation of Cortical Neurons Using Organic Light Emitting Diodes (OLEDs)

2019 ◽  
Author(s):  
Arati Sridharan ◽  
Ankur Shah ◽  
Swathy Sampath Kumar ◽  
James Kyeh ◽  
Joseph Smith ◽  
...  
Keyword(s):  
Mouse Model ◽  
Transgenic Mouse ◽  
Cortical Neurons ◽  
Microelectrode Array ◽  
Light Emitting Diodes ◽  
Green Light ◽  
Transgenic Mouse Model ◽  
Light Emitting

ABSTRACTObjectiveThere is a need for low power, scalable photoelectronic devices and systems for emerging optogenetic needs in neuromodulation. Conventional light emitting diodes (LEDs) are constrained by power and lead-counts necessary for scalability. Organic LEDs (OLEDs) offer an exciting approach to decrease power and lead-counts while achieving high channel counts on thin, flexible substrates that conform to brain surfaces or peripheral neuronal fibers. In this study, we investigate the potential for using OLEDs to modulate neuronal networks cultured in vitro on a transparent microelectrode array (MEA) and subsequently validate neurostimulation in vivo in a transgenic mouse model.ApproachCultured mouse cortical neurons were transfected with light-sensitive opsins such as blue-light sensitive channel-rhodopsin (ChR2) and green-light sensitive chimeric channel-rhodopsin (C1V1tt) and stimulated using blue and green OLEDs (with 455 and 520 nm peak emission spectra respectively) at a power of 1 mW/mm2 under pulsed conditions.Main resultsWe demonstrate neuromodulation and optostimulus-locked, single unit-neuronal activity in neurons expressing stimulating and inhibiting opsins (n=4 MEAs, each with 16 recordable channels). We also validated the optostimulus-locked response in a channel-rhodopsin expressing transgenic mouse model, where at least three isolatable single neuronal cortical units respond to OLED stimulation.SignificanceThe above results indicate the feasibility of generating sufficient luminance from OLEDs to perform neuromodulation both in vitro and in vivo. This opens up the possibility of developing thin, flexible OLED films with multiple stimulation sites that can conform to the shape of the neuronal targets in the brain or the peripheral nervous system. However, stability of these OLEDs under chronic conditions still needs to be carefully assessed with appropriate packaging approaches.

scholarly journals Gut microbiota from multiple sclerosis patients enables spontaneous autoimmune encephalomyelitis in mice

2017 ◽  
Vol 114 (40) ◽  
pp. 10719-10724 ◽  
Author(s):  
Kerstin Berer ◽  
Lisa Ann Gerdes ◽  
Egle Cekanaviciute ◽  
Xiaoming Jia ◽  
Liang Xiao ◽  
...  
Keyword(s):  
Multiple Sclerosis ◽  
Autoimmune Disease ◽  
Mouse Model ◽  
Transgenic Mouse ◽  
Immune Cells ◽  
Disease Incidence ◽  
Transgenic Mouse Model ◽  
Microbial Composition ◽  
Multiple Sclerosis Patients

There is emerging evidence that the commensal microbiota has a role in the pathogenesis of multiple sclerosis (MS), a putative autoimmune disease of the CNS. Here, we compared the gut microbial composition of 34 monozygotic twin pairs discordant for MS. While there were no major differences in the overall microbial profiles, we found a significant increase in some taxa such as Akkermansia in untreated MS twins. Furthermore, most notably, when transplanted to a transgenic mouse model of spontaneous brain autoimmunity, MS twin-derived microbiota induced a significantly higher incidence of autoimmunity than the healthy twin-derived microbiota. The microbial profiles of the colonized mice showed a high intraindividual and remarkable temporal stability with several differences, including Sutterella, an organism shown to induce a protective immunoregulatory profile in vitro. Immune cells from mouse recipients of MS-twin samples produced less IL-10 than immune cells from mice colonized with healthy-twin samples. IL-10 may have a regulatory role in spontaneous CNS autoimmunity, as neutralization of the cytokine in mice colonized with healthy-twin fecal samples increased disease incidence. These findings provide evidence that MS-derived microbiota contain factors that precipitate an MS-like autoimmune disease in a transgenic mouse model. They hence encourage the detailed search for protective and pathogenic microbial components in human MS.

scholarly journals HDAC6/HNF4α loop mediated by miR-1 promotes bile acids-induced gastric intestinal metaplasia

2020 ◽  
Vol 24 (1) ◽  
pp. 103-116
Author(s):  
Na Wang ◽  
Min Chen ◽  
Zhen Ni ◽  
Ting Li ◽  
Jiaoxia Zeng ◽  
...  
Keyword(s):  
Mouse Model ◽  
Intestinal Metaplasia ◽  
Bile Acids ◽  
Transgenic Mouse ◽  
Cell Model ◽  
Critical Role ◽  
Precancerous Lesion ◽  
Transgenic Mouse Model ◽  
Luciferase Reporter ◽  
Gastric Intestinal Metaplasia

Abstract Background Gastric intestinal metaplasia (IM) is considered a precancerous lesion, and bile acids (BA) play a critical role in the induction of IM. Ectopic expression of HNF4α was observed in a BA-induced IM cell model. However, the mechanisms underlying the upregulation of the protein in IM cells remains to be elucidated. Methods The effects of HNF4α on gastric mucosal cells in vivo were identified by a transgenic mouse model and RNA-seq was used to screen downstream targets of deoxycholic acid (DCA). The expression of pivotal molecules and miR-1 was detected by immunohistochemistry and in situ hybridization in normal, gastritis and IM tissue slides or microarrays. The transcriptional regulation of HDAC6 was investigated by chromatin immunoprecipitation (ChIP) and luciferase reporter assays. Results The transgenic mouse model validated that HNF4α stimulated the HDAC6 expression and mucin secretion in gastric mucosa. Increased HDAC6 and HNF4α expression was also detected in the gastric IM cell model and patient specimens. HNF4α could bind to and activate HDAC6 promoter. In turn, HDAC6 enhanced the HNF4α protein level in GES-1 cells. Furthermore, miR-1 suppressed the expression of downstream intestinal markers by targeting HDAC6 and HNF4α. Conclusions Our findings show that the HDAC6/HNF4α loop regulated by miR-1 plays a critical role in gastric IM. Blocking the activation of this loop could be a potential approach to preventing BA-induced gastric IM or even gastric cancer (GC).

scholarly journals SARS-CoV-2 Mpro inhibitors with antiviral activity in a transgenic mouse model

Science ◽  
2021 ◽  
pp. eabf1611
Author(s):  
Jingxin Qiao ◽  
Yue-Shan Li ◽  
Rui Zeng ◽  
Feng-Liang Liu ◽  
Rong-Hua Luo ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Mouse Model ◽  
Transgenic Mouse ◽  
Transgenic Mouse Model ◽  
Global Public Health ◽  
Viral Loads ◽  
Main Protease ◽  
Pharmacokinetic Properties ◽  
Intraperitoneal Treatment

The COVID-19 pandemic caused by the SARS-CoV-2 virus continually poses serious threats to global public health. The main protease (Mpro) of SARS-CoV-2 plays a central role in viral replication. We designed and synthesized 32 new bicycloproline-containing Mpro inhibitors derived from either Boceprevir or Telaprevir, both of which are approved antivirals. All compounds inhibited SARS-CoV-2 Mpro activity in vitro with IC50 values ranging from 7.6 to 748.5 nM. The co-crystal structure of Mpro in complex with MI-23, one of the most potent compounds, revealed its interaction mode. Two compounds (MI-09 and MI-30) showed excellent antiviral activity in cell-based assays. In a SARS-CoV-2 infection transgenic mouse model, oral or intraperitoneal treatment with MI-09 or MI-30 significantly reduced lung viral loads and lung lesions. Both also displayed good pharmacokinetic properties and safety in rats.

scholarly journals Fluorescent indolizine derivative YI-13 detects amyloid-β monomers, dimers, and plaques in the brain of 5XFAD Alzheimer transgenic mouse model

PLoS ONE ◽  
2020 ◽  
Vol 15 (12) ◽  
pp. e0243041
Author(s):  
DaWon Kim ◽  
Jeong Hwa Lee ◽  
Hye Yun Kim ◽  
Jisu Shin ◽  
Kyeonghwan Kim ◽  
...  
Keyword(s):  
Mouse Model ◽  
Transgenic Mouse ◽  
Neurodegenerative Disorder ◽  
Amyloid Β ◽  
Transgenic Mouse Model ◽  
Aβ Oligomers ◽  
Aβ Peptides ◽  
Soluble Aβ Oligomers ◽  
The Brain

Alzheimer disease (AD) is a neurodegenerative disorder characterized by the aberrant production and accumulation of amyloid-β (Aβ) peptides in the brain. Accumulated Aβ in soluble oligomer and insoluble plaque forms are considered to be a pathological culprit and biomarker of the disorder. Here, we report a fluorescent universal Aβ-indicator YI-13, 5-(4-fluorobenzoyl)-7,8-dihydropyrrolo[1,2-b]isoquinolin-9(6H)-one, which detects Aβ monomers, dimers, and plaques. We synthesized a library of 26 fluorescence chemicals with the indolizine core and screen them through a series of in vitro tests utilizing Aβ as a target and YI-13 was selected as the final imaging candidate. YI-13 was found to stain and visualize insoluble Aβ plaques in the brain tissue, of a transgenic mouse model with five familial AD mutations (5XFAD), by a histochemical approach and to label soluble Aβ oligomers within brain lysates of the mouse model under a fluorescence plate reader. Among oligomers aggregated from monomers and synthetic dimers from chemically conjugated monomers, YI-13 preferred the dimeric Aβ.

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