scholarly journals Cell-Type-Specific Circadian Bioluminescence Rhythms in Dbp Reporter Mice

2022 ◽  
pp. 074873042110694
Author(s):  
Ciearra B. Smith ◽  
Vincent van der Vinne ◽  
Eleanor McCartney ◽  
Adam C. Stowie ◽  
Tanya L. Leise ◽  
...  
Keyword(s):  
Locomotor Activity ◽  
Circadian Rhythms ◽  
Ex Vivo ◽  
Luciferase Expression ◽  
Dependent Manner ◽  
Cell Type ◽  
Reporter Mice ◽  
Mouse Tissues ◽  
Cell Type Specific

Circadian rhythms are endogenously generated physiological and molecular rhythms with a cycle length of about 24 h. Bioluminescent reporters have been exceptionally useful for studying circadian rhythms in numerous species. Here, we report development of a reporter mouse generated by modification of a widely expressed and highly rhythmic gene encoding D-site albumin promoter binding protein ( Dbp). In this line of mice, firefly luciferase is expressed from the Dbp locus in a Cre recombinase-dependent manner, allowing assessment of bioluminescence rhythms in specific cellular populations. A mouse line in which luciferase expression was Cre-independent was also generated. The Dbp reporter alleles do not alter Dbp gene expression rhythms in liver or circadian locomotor activity rhythms. In vivo and ex vivo studies show the utility of the reporter alleles for monitoring rhythmicity. Our studies reveal cell-type-specific characteristics of rhythms among neuronal populations within the suprachiasmatic nuclei ex vivo. In vivo studies show Dbp-driven bioluminescence rhythms in the liver of Albumin-Cre;Dbp KI/+ “liver reporter” mice. After a shift of the lighting schedule, locomotor activity achieved the proper phase relationship with the new lighting cycle more rapidly than hepatic bioluminescence did. As previously shown, restricting food access to the daytime altered the phase of hepatic rhythmicity. Our model allowed assessment of the rate of recovery from misalignment once animals were provided with food ad libitum. These studies confirm the previously demonstrated circadian misalignment following environmental perturbations and reveal the utility of this model for minimally invasive, longitudinal monitoring of rhythmicity from specific mouse tissues.

scholarly journals Cell-type specific circadian bioluminescence rhythms recorded from Dbp reporter mice reveal circadian oscillator misalignment

2021 ◽  
Author(s):  
Ciearra B. Smith ◽  
Vincent van der Vinne ◽  
Eleanor McCartney ◽  
Adam C. Stowie ◽  
Tanya L. Leise ◽  
...  
Keyword(s):  
Locomotor Activity ◽  
Circadian Rhythms ◽  
Luciferase Expression ◽  
Dependent Manner ◽  
Cell Type ◽  
Reporter Mice ◽  
Mouse Tissues ◽  
Cell Type Specific

Circadian rhythms are endogenously generated physiological and molecular rhythms with a cycle length of about 24 h. Bioluminescent reporters have been exceptionally useful for studying circadian rhythms in numerous species. Here, we report development of a reporter mouse generated by modification of a widely expressed and highly rhythmic gene encoding D-site albumin promoter binding protein (Dbp). In this line of mice, firefly luciferase is expressed from the Dbp locus in a Cre-recombinase-dependent manner, allowing assessment of bioluminescence rhythms in specific cellular populations. A mouse line in which luciferase expression was Cre-independent was also generated. The Dbp reporter alleles do not alter Dbp gene expression rhythms in liver or circadian locomotor activity rhythms. In vitro and In vivo studies show the utility of the reporter alleles for monitoring rhythmicity. Our studies reveal cell-type specific characteristics of rhythms among neuronal populations within the suprachiasmatic nuclei in vitro. In vivo studies show stable Dbp-driven bioluminescence rhythms in the liver of Albumin-Cre;DbpKI/+ liver reporter mice. After a shift of the lighting schedule, locomotor activity achieved the proper phase relationship with the new lighting cycle more rapidly than hepatic bioluminescence did. As previously shown, restricting food access to the daytime altered the phase of hepatic rhythmicity. Our model allowed assessment of the rate of recovery from misalignment once animals were provided with food ad libitum. These studies provide clear evidence for circadian misalignment following environmental perturbations and reveal the utility of this model for minimally invasive, longitudinal monitoring of rhythmicity from specific mouse tissues.

scholarly journals Genetic engineering for in vivo optical interrogation of neuronal responses to cell type-specific silencing

2021 ◽  
Author(s):  
Firat Terzi ◽  
Johannes Knabbe ◽  
Sidney B. Cambridge
Keyword(s):  
High Resolution ◽  
Genetic Engineering ◽  
Transgene Expression ◽  
Neuronal Morphology ◽  
Dependent Manner ◽  
Cell Type ◽  
Fluorescent Marker ◽  
Genetic Perturbations ◽  
Cell Type Specific

SummaryGenetic engineering of quintuple transgenic brain tissue was used to establish a low background, Cre-dependent version of the inducible Tet-On system for fast, cell type-specific transgene expression in vivo. Co-expression of a constitutive, Cre-dependent fluorescent marker selectively allowed single cell analyses before and after inducible, tet-dependent transgene expression. Here, we used this method for acute, high-resolution manipulation of neuronal activity in the living brain. Single induction of the potassium channel Kir2.1 produced cell type-specific silencing within hours that lasted for at least three days. Longitudinal in vivo imaging of spontaneous calcium transients and neuronal morphology demonstrated that prolonged silencing did not alter spine densities or synaptic input strength. Furthermore, selective induction of Kir2.1 in parvalbumin interneurons increased the activity of surrounding neurons in a distance-dependent manner. This high-resolution, inducible interference and interval imaging of individual cells (high I5, ‘HighFive’) method thus allows visualizing temporally precise, genetic perturbations of defined cells.

scholarly journals Reporter mice for isolating and auditing cell type‐specific extracellular vesicles in vivo

genesis ◽  
2020 ◽  
Vol 58 (7) ◽  
Author(s):  
James V. McCann ◽  
Steven R. Bischoff ◽  
Yu Zhang ◽  
Dale O. Cowley ◽  
Veronica Sanchez‐Gonzalez ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Cell Type ◽  
Reporter Mice ◽  
Cell Type Specific

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 Cell type-specific isolation and transcriptomic profiling informs glial pathology in human temporal lobe epilepsy

2020 ◽  
Author(s):  
Jessica Tome-Garcia ◽  
German Nudelman ◽  
Zarmeen Mussa ◽  
Elodia Caballero ◽  
Yan Jiang ◽  
...  
Keyword(s):  
Temporal Lobe Epilepsy ◽  
Temporal Lobe ◽  
Ex Vivo ◽  
Hybrid Population ◽  
Rna Seq ◽  
Cell Type ◽  
Fresh Frozen ◽  
Cell Type Specific

The pathophysiology of epilepsy underlies complex network dysfunction, the cell-type-specific contributions of which remain poorly defined in human disease. In this study, we developed a strategy that simultaneously isolates neuronal, astrocyte and oligodendroglial progenitor (OPC)-enriched nuclei from human fresh-frozen neocortex and applied it to characterize the distinct transcriptome of each cell type in temporal lobe epilepsy (TLE) surgical samples. Differential RNA-seq analysis revealed several dysregulated pathways in neurons, OPCs, and astrocytes, and disclosed an immature phenotype switch in TLE astrocytes. An independent single cell RNA-seq TLE dataset uncovered a hybrid population of cells aberrantly co-expressing canonical astrocyte and OPC-like progenitor markers (GFAP+OLIG2+ glia), which we corroborated in-situ in human TLE samples, and further demonstrated their emergence after chronic seizure injury in a mouse model of status epilepticus. In line with their immature signature, a subset of human TLE glia were also abnormally proliferative, both in-vivo and in-vitro. Generally, this analysis validates the utility of the proposed cell type-specific isolation strategy to study glia-specific changes ex vivo using fresh-frozen human samples, and specifically, it delineates an aberrant glial phenotype in human TLE specimens.

scholarly journals Genetic analysis reveals cell type-specific regulation of receptor tyrosine kinase c-Kit by the protein tyrosine phosphatase SHP1.

1996 ◽  
Vol 184 (3) ◽  
pp. 1111-1126 ◽  
Author(s):  
U Lorenz ◽  
A D Bergemann ◽  
H N Steinberg ◽  
J G Flanagan ◽  
X Li ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mast Cells ◽  
Genetic Analysis ◽  
Progenitor Cells ◽  
Ex Vivo ◽  
Receptor Protein ◽  
Cell Type ◽  
Protein Tyrosine ◽  
Cell Type Specific

Receptor protein tyrosine kinases (RTKs) transmit downstream signals via interactions with secondary signaling molecules containing SH2 domains. Although many SH2-phosphotyrosyl interactions have been defined in vitro, little is known about the physiological significance of specific RTK/SH2 interactions in vivo. Also, little is known about the mechanisms by which specific RTKs interact with and/or are regulated by specific protein tyrosine phosphatases (PTPs). To address such issue, we carried out a genetic analysis of the previously reported biochemical interaction between the RTK c-Kit, encoded at the W locus, and the SH2-containing non-transmembrane PTP SHP1, encoded at the motheaten (me) locus (1). Mice carrying a kinase-defective allele of c-Kit (Wv/+) were crossed with me/+ mice, which carry one effectively null allele of SHP1, and then backcrossed to generate all possible allelic combinations. Our results indicate strong intergenic complementation between these loci in hematopoietic progenitor cells. Compared to progenitors purified from normal mice, bone marrow progenitor cells (lin-) from me/me mice markedly hyper-proliferated in response to Kit ligand (KL). stimulation. Superimposition of the me/me genotype increased the number of one marrow-derived CFU-E from Wv/+ mice. Conversely, the presence of one or two copies of Wv decreased the number of macrophages and granulocytes in me/me lung, skin, peripheral blood and bone marrow, thereby decreasing the severity of the me/me phenotype. The decrease in dermal mast cells in Wv/Wv mice was rescued to levels found in Wv/+mice by superimposition of the me/me genotype. Surprisingly, however, the presence or absence of SHP1 had no effect on the proliferative response of bone marrow-derived cultured mast cells to KL or IL3 ex vivo. Nevertheless, the immediate-early response to KL stimulation, as measured by KL-induced tyrosyl phosphorylation, was substantially increased in mast cells from Wv/+:me/me compared to Wv/ +:+/+ mice, strongly suggesting that SHP1 directly dephosphorylates and regulates c-Kit. Taken together, our results establish that SHP1 negatively regulates signaling from c-Kit in vivo, but in a cell type-specific manner.

ITag-RNA Allows in Vivo Cell-Type Specific Transcriptional Characterization and Tracking of Circulating Transcripts

2018 ◽  
Author(s):  
J. Darr ◽  
M. Lassi ◽  
R. Gerlini ◽  
F. Scheid ◽  
M. Hrabě de Angelis ◽  
...  
Keyword(s):  
Cell Type ◽  
Cell Type Specific

Ethanol Extract of Achillea fragrantissima Enhances Angiogenesis through Stimulation of VEGF Production

2020 ◽  
Vol 21 ◽  
Author(s):  
Hana M. Hammad ◽  
Amer Imraish ◽  
Maysa Al-Hussaini ◽  
Malek Zihlif ◽  
Amani A. Harb ◽  
...  
Keyword(s):  
Wound Healing ◽  
Rural Communities ◽  
Ex Vivo ◽  
Ethanol Extract ◽  
Aortic Ring ◽  
Treated Group ◽  
Control Group ◽  
Dependent Manner ◽  
Achillea Fragrantissima

Objective: Achillea fragrantissima L. (Asteraceae) is a traditionally used medicinal herb in the rural communities of Jordan. Methods: The present study evaluated the efficacy of the ethanol extract of this species on angiogenesis in both, ex vivo using rat aortic ring assay and in vivo using rat excision wound model. Results: In concentrations of 50 and 100 µg/ml, the ethanol extract showed angiogenic stimulatory effect and significantly increased length of capillary protrusions around aorta rings of about 60% in comparison to those of untreated aorta rings. In MCF-7 cells, the ethanol extract of A. fragrantissima stimulates the production of VEGF in a dose-dependent manner. 1% and 5% of ethanol extract of A. fragrantissima containing vaseline based ointment was applied on rat excision wounds for six days and was found to be effective in wound healing and maturation of the scar. Both preparations resulted in better wound healing when compared to the untreated control group and vaseline-treated group. This effect was comparable to that induced by MEBO, the positive control. Conclusion: The results indicate that A. fragrantissima has a pro-angiogenic effect, which may act through the VEGF signaling pathway.

Differential pial and penetrating arterial responses examined by optogenetic activation of astrocytes and neurons

2021 ◽  
pp. 0271678X2110103
Author(s):  
Nao Hatakeyama ◽  
Miyuki Unekawa ◽  
Juri Murata ◽  
Yutaka Tomita ◽  
Norihiro Suzuki ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Acetylcholine Receptors ◽  
Step Function ◽  
Muscarinic Acetylcholine Receptors ◽  
Cell Type ◽  
Function Type ◽  
Neuron Activation ◽  
Cell Type Specific ◽  
Arterial Responses

A variety of brain cells participates in neurovascular coupling by transmitting and modulating vasoactive signals. The present study aimed to probe cell type-dependent cerebrovascular (i.e., pial and penetrating arterial) responses with optogenetics in the cortex of anesthetized mice. Two lines of the transgenic mice expressing a step function type of light-gated cation channel (channelrhodopsine-2; ChR2) in either cortical neurons (muscarinic acetylcholine receptors) or astrocytes (Mlc1-positive) were used in the experiments. Photo-activation of ChR2-expressing astrocytes resulted in a widespread increase in cerebral blood flow (CBF), extending to the nonstimulated periphery. In contrast, photo-activation of ChR2-expressing neurons led to a relatively localized increase in CBF. The differences in the spatial extent of the CBF responses are potentially explained by differences in the involvement of the vascular compartments. In vivo imaging of the cerebrovascular responses revealed that ChR2-expressing astrocyte activation led to the dilation of both pial and penetrating arteries, whereas ChR2-expressing neuron activation predominantly caused dilation of the penetrating arterioles. Pharmacological studies showed that cell type-specific signaling mechanisms participate in the optogenetically induced cerebrovascular responses. In conclusion, pial and penetrating arterial vasodilation were differentially evoked by ChR2-expressing astrocytes and neurons.

scholarly journals Uptake and Distribution of Administered Bone Marrow Mesenchymal Stem Cell Extracellular Vesicles in Retina

Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 730
Author(s):  
Biji Mathew ◽  
Leianne A. Torres ◽  
Lorea Gamboa Gamboa Acha ◽  
Sophie Tran ◽  
Alice Liu ◽  
...  
Keyword(s):  
Stem Cell ◽  
Mesenchymal Stem Cell ◽  
Extracellular Vesicles ◽  
Time Course ◽  
Ganglion Cells ◽  
Ex Vivo ◽  
Dependent Manner ◽  
Paracrine Effects

Cell replacement therapy using mesenchymal (MSC) and other stem cells has been evaluated for diabetic retinopathy and glaucoma. This approach has significant limitations, including few cells integrated, aberrant growth, and surgical complications. Mesenchymal Stem Cell Exosomes/Extracellular Vesicles (MSC EVs), which include exosomes and microvesicles, are an emerging alternative, promoting immunomodulation, repair, and regeneration by mediating MSC’s paracrine effects. For the clinical translation of EV therapy, it is important to determine the cellular destination and time course of EV uptake in the retina following administration. Here, we tested the cellular fate of EVs using in vivo rat retinas, ex vivo retinal explant, and primary retinal cells. Intravitreally administered fluorescent EVs were rapidly cleared from the vitreous. Retinal ganglion cells (RGCs) had maximal EV fluorescence at 14 days post administration, and microglia at 7 days. Both in vivo and in the explant model, most EVs were no deeper than the inner nuclear layer. Retinal astrocytes, microglia, and mixed neurons in vitro endocytosed EVs in a dose-dependent manner. Thus, our results indicate that intravitreal EVs are suited for the treatment of retinal diseases affecting the inner retina. Modification of the EV surface should be considered for maintaining EVs in the vitreous for prolonged delivery.

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