scholarly journals Luciferase Reporter Mice for In Vivo Monitoring and Ex Vivo Assessment of Hypothalamic Signaling of Socs3 Expression

2019 ◽  
Vol 3 (7) ◽  
pp. 1246-1260
Author(s):  
Elizabeth L Cordonier ◽  
Tiemin Liu ◽  
Kenji Saito ◽  
Siyu S Chen ◽  
Yong Xu ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Luciferase Reporter ◽  
In Vivo Monitoring ◽  
Reporter Mice ◽  
Socs3 Expression

scholarly journals Bioluminescent Detection of Endotoxin Effects on HIV-1 LTR-driven Transcription in Vivo

2003 ◽  
Vol 51 (6) ◽  
pp. 741-749 ◽  
Author(s):  
Fiona E. Yull ◽  
Wei Han ◽  
E. Duco Jansen ◽  
M. Brett Everhart ◽  
Ruxana T. Sadikot ◽  
...  
Keyword(s):  
Bioluminescence Imaging ◽  
Luciferase Activity ◽  
Ex Vivo ◽  
Photon Emission ◽  
Luciferase Assay ◽  
Luciferase Reporter ◽  
Luciferase Expression ◽  
Reporter Mice ◽  
Hiv 1

We investigated the effects of Gram-negative bacterial lipopolysaccharide (LPS) on luciferase expression in transgenic reporter mice in which luciferase expression is driven by the nuclear factor κB (NF-κB)-dependent portion of the human immunodeficiency virus-1 (HIV-1) long terminal repeat (HIV-1 LTR). Using these mice, we dissected the sources of luciferase activity at the organ level by (a) assessing luciferase activity in organ homogenates, (b) bioluminescence imaging in vivo, and (c) bioluminescence imaging of individual organs ex vivo. Luciferin dosage was a critical determinant of the magnitude of photon emission from these reporter mice. Photon emission increased at doses from 0.5–6 mg of luciferin given by intraperitoneal (IP) injection. The differential between basal and LPS-induced bioluminescence was maximal at 3–6 mg of luciferin. Luciferase expression was highly inducible in lungs, liver, spleen, and kidneys after a single IP injection of LPS, as assessed by luciferase activity measurements in organ homogenates. Luciferase activity was also induced in the forebrain by treatment with IP LPS. In contrast, aerosolized LPS produced a response localized to the lungs as assessed by both bioluminescence and ex vivo luciferase assay measurements. These studies demonstrate the utility of luciferase reporter mice for determining organ-specific gene expression in response to local and systemic stimuli.

scholarly journals Correction: Knock-in Luciferase Reporter Mice for In Vivo Monitoring of CREB Activity

PLoS ONE ◽  
2016 ◽  
Vol 11 (9) ◽  
pp. e0163576
Author(s):  
Dmitry Akhmedov ◽  
Kavitha Rajendran ◽  
Maria G. Mendoza-Rodriguez ◽  
Rebecca Berdeaux
Keyword(s):  
Luciferase Reporter ◽  
In Vivo Monitoring ◽  
Reporter Mice

scholarly journals Knock-in Luciferase Reporter Mice for In Vivo Monitoring of CREB Activity

PLoS ONE ◽  
2016 ◽  
Vol 11 (6) ◽  
pp. e0158274 ◽  
Author(s):  
Dmitry Akhmedov ◽  
Kavitha Rajendran ◽  
Maria G. Mendoza-Rodriguez ◽  
Rebecca Berdeaux
Keyword(s):  
Luciferase Reporter ◽  
In Vivo Monitoring ◽  
Reporter Mice

Abstract 074: GRK5 Increases Cardiac NFAT Transcriptional Activity

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Jonathan Hullmann ◽  
J K Chuprun ◽  
Erhe Gao ◽  
Walter J Koch
Keyword(s):  
Cardiac Hypertrophy ◽  
Ex Vivo ◽  
Pressure Overload ◽  
Luciferase Reporter ◽  
Wild Type ◽  
Activated T Cells ◽  
Reporter Mice ◽  
Nuclear Dependence ◽  
Independent Effects

Introduction: G protein-coupled receptor (GPCR) kinase-2 and -5 (GRK2 and GRK5) have been shown to be up-regulated in failing human myocardium. While the canonical role of GRKs is to desensitize receptors via phosphorylation, it has been shown that GRK5, unlike GRK2, can reside in the nucleus of cardiomyocytes and exert GPCR-independent effects that promote maladaptive cardiac hypertrophy and heart failure (HF). Previously, our lab has shown that GRK5 increases hypertrophic gene transcription through the phosphorylation of histone deacetylase 5 (HDAC5) and subsequent disinhibition of the transcription factor, myocyte enhancer factor-2 (MEF2). Use of GRK5 knockout (KO) mice has recently proved that GRK5 is indeed a physiological HDAC kinase during hypertrophic stress. Through experiments described below we have now identified the nuclear factor of activated T cells (NFAT) pathway as another potential target of GRK5 during cardiac hypertrophy that may play a role in its facilitation of HF. Methods and Results: Cardiac-specific NFAT-luciferase reporter mice were crossed with mice that overexpress wild-type GRK5 in myocytes to determine the role for GRK5 in NFAT signaling. These double-transgenic mice along with controls were stressed using the hypertrophic α 1 -adrenergic agonist phenylephrine (PE) as well as ventricular pressure-overload via transverse aortic constriction (TAC). NFAT activity was determined by both in vivo and ex vivo luciferase assays as well as RT-PCR for the NFAT target gene RCAN. Cardiac specific GRK5 overexpression leads to an increase in NFAT activity in the basal state as well as after both forms of hypertrophic stress. This was reproduced in cultured myocytes using a NFAT-reporter construct. Overexpression of a mutant GRK5 that cannot enter the nucleus appears to not activate NFAT demonstrating a nuclear-dependence. Consistent with this, GRK5 KO mice after TAC showed a decrease in the up-regulation of RCAN transcription as compared to wild-type mice. Studies are ongoing to determine the mechanism of GRK5’s regulation of cardiac NFAT activity. Conclusions: This study provides another non-canonical role for GRK5 in activating the hypertrophic NFAT pathway in cardiomyocytes.

Abstract P466: Circadian Rhythm Disruptions in a Novel Diabetic db/db-mPer2 Luc Mouse Model

Hypertension ◽  
2017 ◽  
Vol 70 (suppl_1) ◽  
Author(s):  
Tianfei Hou ◽  
Wen Su ◽  
Ming C Gong ◽  
Zhenheng Guo
Keyword(s):  
Blood Pressure ◽  
Circadian Rhythms ◽  
Real Time ◽  
Clock Gene ◽  
Ex Vivo ◽  
Phase Advance ◽  
Luciferase Reporter ◽  
High Time Resolution ◽  
Peripheral Tissues

Db/db mouse, which lacks functional leptin receptor, is an extensively used model of obesity and type 2 diabetes. We and others have demonstrated that db/db mouse has disruptions in circadian rhythms of behavior, physiology and some clock genes. However, systemic investigations of the alterations in clock gene oscillations in multiple systems with high time resolution in this model are impeded by the impractical demand for large number of animals. To overcome this limitation, we cross bred the db/db mouse with mPer2 Luc mouse in which the clock gene Period2 is fused with a luciferase reporter thus allow real-time monitoring of the clock gene Per2 oscillations. The generated db/db-mPer2 Luc mice had the typical diabetic mellitus including obesity, hyperglycemia, hyperinsulinemia, glucose intolerance and insulin resistance. In addition, the db/db-mPer2 Luc mice also exhibited disruptions in circadian rhythms in behavior (locomotor activity), physiology (blood pressure) and metabolism (respiratory exchange ratio and energy expenditure). Using the LumiCycle system, we monitored in real-time of the Per2 oscillations in both the SCN central clock and multiple peripheral tissues ex vivo . The results showed no difference in the phase of the central SCN Per2 oscillation. However, the peripheral tissues that related to metabolism, such as liver and white adipose clocks, displayed 3.28±0.86 and 4.64±1.06 hours of phase advance respectively. Aorta, mesentery artery and kidney, organs play important role in blood pressure homeostasis, showed 0.99±0.37, and 2.12±0.4, and 2.21±0.5 hours phase advance respectively. Interestingly, no difference was observed in the lung and adrenal gland. We then investigated the Per2 oscillation in vivo by using the IVIS imaging system. Consistent with the ex vivo results, the liver Per2 oscillation were phase advanced in vivo. Our findings demonstrated that clock gene Per2 oscillations were disrupted in multiple peripheral tissues but not in central SCN. Moreover, the extent of phase advance in peripheral tissue varies largely. Our results suggest dyssynchrony of the clock oscillations among various peripheral systems likely contribute to the multiple disruptions in physiology and metabolism in diabetic db/db mice.

scholarly journals Mast cells acquire MHCII from dendritic cells during skin inflammation

2017 ◽  
Vol 214 (12) ◽  
pp. 3791-3811 ◽  
Author(s):  
Jan Dudeck ◽  
Anna Medyukhina ◽  
Julia Fröbel ◽  
Carl-Magnus Svensson ◽  
Johanna Kotrba ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
T Cell ◽  
Mast Cells ◽  
Ex Vivo ◽  
Multiphoton Microscopy ◽  
Skin Inflammation ◽  
Critical Periods ◽  
Reporter Mice ◽  
Antigen Presenting

Mast cells (MCs) and dendritic cells (DCs) are essential innate sentinels populating host-environment interfaces. Using longitudinal intravital multiphoton microscopy of DCGFP/MCRFP reporter mice, we herein provide in vivo evidence that migratory DCs execute targeted cell-to-cell interactions with stationary MCs before leaving the inflamed skin to draining lymph nodes. During initial stages of skin inflammation, DCs dynamically scan MCs, whereas at a later stage, long-lasting interactions predominate. These innate-to-innate synapse-like contacts ultimately culminate in DC-to-MC molecule transfers including major histocompatibility complex class II (MHCII) proteins enabling subsequent ex vivo priming of allogeneic T cells with a specific cytokine signature. The extent of MHCII transfer to MCs correlates with their T cell priming efficiency. Importantly, preventing the cross talk by preceding DC depletion decreases MC antigen presenting capacity and T cell–driven inflammation. Consequently, we identify an innate intercellular communication arming resident MCs with key DC functions that might contribute to the acute defense potential during critical periods of migration-based DC absence.

scholarly journals IL-4–secreting eosinophils promote endometrial stromal cell proliferation and preventChlamydia-induced upper genital tract damage

2017 ◽  
Vol 114 (33) ◽  
pp. E6892-E6901 ◽  
Author(s):  
Rodolfo D. Vicetti Miguel ◽  
Nirk E. Quispe Calla ◽  
Darlene Dixon ◽  
Robert A. Foster ◽  
Andrea Gambotto ◽  
...  
Keyword(s):  
Genital Tract ◽  
Tissue Repair ◽  
Stromal Cell ◽  
Immune Cell ◽  
Ex Vivo ◽  
Endometrial Stromal Cell ◽  
Wild Type ◽  
Reporter Mice

GenitalChlamydia trachomatisinfections in women typically are asymptomatic and do not cause permanent upper genital tract (UGT) damage. Consistent with this presentation, type 2 innate and TH2 adaptive immune responses associated with dampened inflammation and tissue repair are elicited in the UGT ofChlamydia-infected women. PrimaryC. trachomatisinfection of mice also causes no genital pathology, but unlike women, does not generateChlamydia-specific TH2 immunity. Herein, we explored the significance of type 2 innate immunity for restricting UGT tissue damage inChlamydia-infected mice, and in initial studies intravaginally infected wild-type, IL-10−/−, IL-4−/−, and IL-4Rα−/−mice with low-doseC. trachomatisinoculums. WhereasChlamydiawas comparably cleared in all groups, IL-4−/−and IL-4Rα−/−mice displayed endometrial damage not seen in wild-type or IL-10−/−mice. Congruent with the aberrant tissue repair in mice with deficient IL-4 signaling, we found that IL-4Rα and STAT6 signaling mediated IL-4–induced endometrial stromal cell (ESC) proliferation ex vivo, and that genital administration of an IL-4–expressing adenoviral vector greatly increased in vivo ESC proliferation. Studies with IL-4-IRES-eGFP (4get) reporter mice showed eosinophils were the main IL-4–producing endometrial leukocyte (constitutively and duringChlamydiainfection), whereas studies with eosinophil-deficient mice identified this innate immune cell as essential for endometrial repair duringChlamydiainfection. Together, our studies reveal IL-4–producing eosinophils stimulate ESC proliferation and preventChlamydia-induced endometrial damage. Based on these results, it seems possible that the robust type 2 immunity elicited byChlamydiainfection of human genital tissue may analogously promote repair processes that reduce phenotypic disease expression.

scholarly journals A candidate causal variant underlying both higher intelligence and increased risk of bipolar disorder

2019 ◽  
Author(s):  
Susan Q. Shen ◽  
Jeong Sook Kim-Han ◽  
Lin Cheng ◽  
Duo Xu ◽  
Omer Gokcumen ◽  
...  
Keyword(s):  
Bipolar Disorder ◽  
Molecular Mechanisms ◽  
Ex Vivo ◽  
Fetal Brain ◽  
Causal Variant ◽  
Enhancer Activity ◽  
Embryonic Mouse ◽  
Reporter Mice ◽  
Increased Risk

ABSTRACTBipolar disorder is a highly heritable mental illness, but the relevant genetic variants and molecular mechanisms are largely unknown. Recent GWAS’s have identified an intergenic region associated with both intelligence and bipolar disorder. This region contains dozens of putative fetal brain-specific enhancers and is located ~0.7 Mb upstream of the neuronal transcription factor POU3F2. We identified a candidate causal variant, rs77910749, that falls within a highly conserved putative enhancer, LC1. This human-specific variant is a single-base deletion in a PAX6 binding site and is predicted to be functional. We hypothesized that rs77910749 alters LC1 activity and hence POU3F2 expression during neurodevelopment. Indeed, transgenic reporter mice demonstrated LC1 activity in the developing cerebral cortex and amygdala. Furthermore, ex vivo reporter assays in embryonic mouse brain and human iPSC-derived cerebral organoids revealed increased enhancer activity conferred by the variant. To probe the in vivo function of LC1, we deleted the orthologous mouse region, which resulted in amygdala-specific changes in Pou3f2 expression. Lastly, ‘humanized’ rs77910749 knock-in mice displayed behavioral defects in sensory gating, an amygdala-dependent endophenotype seen in patients with bipolar disorder. Our study elucidates a molecular mechanism underlying the long-speculated link between higher cognition and neuropsychiatric disease.

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