scholarly journals Establishment of a Reproducible Ischemic Stroke Model in Nestin-GFP Mice with High Survival Rates

2021 ◽  
Vol 22 (23) ◽  
pp. 12997
Author(s):  
Hideaki Nishie ◽  
Akiko Nakano-Doi ◽  
Toshinori Sawano ◽  
Takayuki Nakagomi
Keyword(s):  
Ischemic Stroke ◽  
Blood Vessels ◽  
Fluorescent Protein ◽  
Expression Patterns ◽  
Yellow Fluorescent Protein ◽  
Survival Rates ◽  
High Survival ◽  
Wild Type ◽  
Mca Occlusion

An accumulation of evidence shows that endogenous neural stem/progenitor cells (NSPCs) are activated following brain injury such as that suffered during ischemic stroke. To understand the expression patterns of these cells, researchers have developed mice that express an NSPC marker, Nestin, which is detectable by specific reporters such as green fluorescent protein (GFP), i.e., Nestin-GFP mice. However, the genetic background of most transgenic mice, including Nestin-GFP mice, comes from the C57BL/6 strain. Because mice from this background strain have many cerebral arterial branches and collateral vessels, they are accompanied by several major problems including variable ischemic areas and high mortality when subjected to ischemic stroke by occluding the middle cerebral artery (MCA). In contrast, CB-17 wild-type mice are free from these problems. Therefore, with the aim of overcoming the aforementioned defects, we first crossed Nestin-GFP mice (C57BL/6 background) with CB-17 wild-type mice and then developed Nestin-GFP mice (CB-17 background) by further backcrossing the generated hybrid mice with CB-17 wild-type mice. Subsequently, we investigated the phenotypes of the established Nestin-GFP mice (CB-17 background) following MCA occlusion; these mice had fewer blood vessels around the MCA compared with the number of blood vessels in Nestin-GFP mice (C57BL/6 background). In addition, TTC staining showed that infarcted volume was variable in Nestin-GFP mice (C57BL/6 background) but highly reproducible in Nestin-GFP mice (CB-17 background). In a further investigation of mice survival rates up to 28 days after MCA occlusion, all Nestin-GFP mice (CB-17 background) survived the period, whereas Nestin-GFP mice (C57BL/6 background) frequently died within 1 week and exhibited a higher mortality rate. Immunohistochemistry analysis of Nestin-GFP mice (CB-17 background) showed that GFP+ cells were mainly obverted in not only conventional neurogenic areas, including the subventricular zone (SVZ), but also ischemic areas. In vitro, cells isolated from the ischemic areas and the SVZ formed GFP+ neurosphere-like cell clusters that gave rise to various neural lineages including neurons, astrocytes, and oligodendrocytes. However, microarray analysis of these cells and genetic mapping experiments by Nestin-CreERT2 Line4 mice crossed with yellow fluorescent protein (YFP) reporter mice (Nestin promoter-driven YFP-expressing mice) indicated that cells with NSPC activities in the ischemic areas and the SVZ had different characteristics and origins. These results show that the expression patterns and fate of GFP+ cells with NSPC activities can be precisely investigated over a long period in Nestin-GFP mice (CB-17 background), which is not necessarily possible with Nestin-GFP mice (C57BL/6 background). Thus, Nestin-GFP mice (CB-17 background) could become a useful tool with which to investigate the mechanism of neurogenesis via the aforementioned cells under pathological conditions such as following ischemic stroke.

scholarly journals Interaction of BAG1 and Hsp70 Mediates Neuroprotectivity and Increases Chaperone Activity

2005 ◽  
Vol 25 (9) ◽  
pp. 3715-3725 ◽  
Author(s):  
Jan Liman ◽  
Sundar Ganesan ◽  
Christoph P. Dohm ◽  
Stan Krajewski ◽  
John C. Reed ◽  
...  
Keyword(s):  
Neuronal Differentiation ◽  
Heat Shock Protein 70 ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Full Length ◽  
Wild Type ◽  
Binding Characteristics ◽  
In Cells

ABSTRACT It was recently shown that Bcl-2-associated athanogene 1 (BAG1) is a potent neuroprotectant as well as a marker of neuronal differentiation. Since there appears to exist an equilibrium within the cell between BAG1 binding to heat shock protein 70 (Hsp70) and BAG1 binding to Raf-1 kinase, we hypothesized that changing BAG1 binding characteristics might significantly alter BAG1 function. To this end, we compared rat CSM14.1 cells and human SHSY-5Y cells stably overexpressing full-length BAG1 or a deletion mutant (BAGΔC) no longer capable of binding to Hsp70. Using a novel yellow fluorescent protein-based foldase biosensor, we demonstrated an upregulation of chaperone in situ activity in cells overexpressing full-length BAG1 but not in cells overexpressing BAGΔC compared to wild-type cells. Interestingly, in contrast to the nuclear and cytosolic localizations of full-length BAG1, BAGΔC was expressed exclusively in the cytosol. Furthermore, cells expressing BAGΔC were no longer protected against cell death. However, they still showed accelerated neuronal differentiation. Together, these results suggest that BAG1-induced activation of Hsp70 is important for neuroprotectivity, while BAG1-dependent modulation of neuronal differentiation in vitro is not.

scholarly journals Candida albicans SNO1 and SNZ1 expressed in stationary-phase planktonic yeast cells and base of biofilm

Microbiology ◽  
2006 ◽  
Vol 152 (7) ◽  
pp. 2031-2038 ◽  
Author(s):  
Priya Uppuluri ◽  
Bhaskarjyoti Sarmah ◽  
W. LaJean Chaffin
Keyword(s):  
Candida Albicans ◽  
Stationary Phase ◽  
Fluorescent Protein ◽  
Protein Localization ◽  
Expression Patterns ◽  
Yellow Fluorescent Protein ◽  
Growth Conditions ◽  
Yeast Cells ◽  
Yeast Culture

The Candida albicans homologues of the most studied Saccharomyces cerevisiae stationary-phase genes, SNO1 and SNZ1, were used to test the hypothesis that, within a biofilm, some cells reach stationary phase within continuously fed, as well as static, C. albicans biofilms grown on dental acrylic. The authors first studied the expression patterns of these two genes in planktonic growth conditions. Using real-time RT-PCR (RT-RTPCR), increased peak expression of both SNZ1 and SNO1 was observed at 5 and 6 days, respectively, in C. albicans grown in suspension culture. SNZ1–yellow fluorescent protein (YFP) and SNO1–YFP were constructed to study expression at the cellular level and protein localization in C. albicans. Snz1p–YFP and Sno1p–YFP localized to the cytoplasm with maximum expression (>90 %) at 5 and 6 days, respectively, in planktonic conditions. When yeast growth was reinitiated, loss of fluorescence began immediately. Germ tubes and hyphae were non-fluorescent. Pseudohyphae began appearing at 9 days in planktonic yeast culture and expressed each protein by 11 days; however, the cells budding from pseudohyphae were not fluorescent. Biofilm was formed in vitro under either static or continuously fed conditions. Increased expression of the two genes was shown by RT-RTPCR, beginning by day 3 and increasing through to day 15 (continuously fed biofilm). Only the bottommost layer of acrylic-adhered cells in the biofilm showed 25 and 40 % fluorescence at 6 and 15 days, respectively. These observations suggest that only a few cells in C. albicans biofilms express genes associated with the planktonic stationary phase and that these are found at the bottom of the biofilm adhered to the surface.

scholarly journals The CRISPR/Cas9 Minipig—A Transgenic Minipig to Produce Specific Mutations in Designated Tissues

Cancers ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 3024
Author(s):  
Martin Fogtmann Berthelsen ◽  
Maria Riedel ◽  
Huiqiang Cai ◽  
Søren H. Skaarup ◽  
Aage K. O. Alstrup ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Somatic Cells ◽  
Genetic Alterations ◽  
Lung Epithelial Cells ◽  
Target Cells ◽  
Multiple Gene ◽  
Conditional Expression ◽  
Gene Alterations

The generation of large transgenic animals is impeded by complex cloning, long maturation and gastrulation times. An introduction of multiple gene alterations increases the complexity. We have cloned a transgenic Cas9 minipig to introduce multiple mutations by CRISPR in somatic cells. Transgenic Cas9 pigs were generated by somatic cell nuclear transfer and were backcrossed to Göttingen Minipigs for two generations. Cas9 expression was controlled by FlpO-mediated recombination and was visualized by translation from red to yellow fluorescent protein. In vitro analyses in primary fibroblasts, keratinocytes and lung epithelial cells confirmed the genetic alterations executed by the viral delivery of single guide RNAs (sgRNA) to the target cells. Moreover, multiple gene alterations could be introduced simultaneously in a cell by viral delivery of sgRNAs. Cells with loss of TP53, PTEN and gain-of-function mutation in KRASG12D showed increased proliferation, confirming a transformation of the primary cells. An in vivo activation of Cas9 expression could be induced by viral delivery to the skin. Overall, we have generated a minipig with conditional expression of Cas9, where multiple gene alterations can be introduced to somatic cells by viral delivery of sgRNA. The development of a transgenic Cas9 minipig facilitates the creation of complex pre-clinical models for cancer research.

Identity of the renin cell is mediated by cAMP and chromatin remodeling: an in vitro model for studying cell recruitment and plasticity

2008 ◽  
Vol 294 (2) ◽  
pp. H699-H707 ◽  
Author(s):  
Ellen Steward Pentz ◽  
Maria Luisa S. Sequeira Lopez ◽  
Magali Cordaillat ◽  
R. Ariel Gomez
Keyword(s):  
Chromatin Remodeling ◽  
In Vitro Model ◽  
Molecular Mechanisms ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Histone H4 Acetylation ◽  
Renin Gene ◽  
Vitro Model

The renin-angiotensin system (RAS) regulates blood pressure and fluid-electrolyte homeostasis. A key step in the RAS cascade is the regulation of renin synthesis and release by the kidney. We and others have shown that a major mechanism to control renin availability is the regulation of the number of cells capable of making renin. The kidney possesses a pool of cells, mainly in its vasculature but also in the glomeruli, capable of switching from smooth muscle to endocrine renin-producing cells when homeostasis is threatened. The molecular mechanisms governing the ability of these cells to turn the renin phenotype on and off have been very difficult to study in vivo. We, therefore, developed an in vitro model in which cells of the renin lineage are labeled with cyan fluorescent protein and cells actively making renin mRNA are labeled with yellow fluorescent protein. The model allowed us to determine that it is possible to culture cells of the renin lineage for numerous passages and that the memory to express the renin gene is maintained in culture and can be reenacted by cAMP and chromatin remodeling (histone H4 acetylation) at the cAMP-responsive element in the renin gene.

scholarly journals tdLanYFP, a yellow, bright, photostable and pH insensitive fluorescent protein for live cell imaging and FRET-based sensing strategies

2021 ◽  
Author(s):  
Y. Bousmah ◽  
H. Valenta ◽  
G. Bertolin ◽  
U. Singh ◽  
V. Nicolas ◽  
...  
Keyword(s):  
Single Molecule ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Resonance Energy ◽  
Live Cell ◽  
Live Cells

AbstractYellow fluorescent proteins (YFP) are widely used as optical reporters in Förster Resonance Energy Transfer (FRET) based biosensors. Although great improvements have been done, the sensitivity of the biosensors is still limited by the low photostability and the poor fluorescence performances of YFPs at acidic pHs. In fact, today, there is no yellow variant derived from the EYFP with a pK1/2 below ∼5.5. Here, we characterize a new yellow fluorescent protein, tdLanYFP, derived from the tetrameric protein from the cephalochordate B. lanceolatum, LanYFP. With a quantum yield of 0.92 and an extinction coefficient of 133 000 mol−1.L.cm−1, it is, to our knowledge, the brightest dimeric fluorescent protein available, and brighter than most of the monomeric YFPs. Contrasting with EYFP and its derivatives, tdLanYFP has a very high photostability in vitro and preserves this property in live cells. As a consequence, tdLanYFP allows the imaging of cellular structures with sub-diffraction resolution with STED nanoscopy. We also demonstrate that the combination of high brightness and strong photostability is compatible with the use of spectro-microscopies in single molecule regimes. Its very low pK1/2 of 3.9 makes tdLanYFP an excellent tag even at acidic pHs. Finally, we show that tdLanYFP can be a FRET partner either as donor or acceptor in different biosensing modalities. Altogether, these assets make tdLanYFPa very attractive yellow fluorescent protein for long-term or single-molecule live-cell imaging that is also suitable for FRET experiment including at acidic pH.

scholarly journals A hyperthermoactive-Cas9 editing tool reveals the role of a unique arsenite methyltransferase in the arsenic resistance system of Thermus thermophilus HB27

2021 ◽  
Author(s):  
Giovanni Gallo ◽  
Ioannis Mougiakos ◽  
Mauricio Bianco ◽  
Miriam Carbonaro ◽  
Andrea Carpentieri ◽  
...  
Keyword(s):  
Catalytic Mechanism ◽  
Fluorescent Protein ◽  
Efflux Pump ◽  
Thermus Thermophilus ◽  
Yellow Fluorescent Protein ◽  
Arsenic Resistance ◽  
Wide Range ◽  
Resistance System

Arsenic detoxification systems can be found in a wide range of organisms, from bacteria to man. In a previous study, we discovered an arsenic-responsive transcriptional regulator in the thermophilic bacterium Thermus thermophilus HB27 (TtSmtB). Here, we characterize the arsenic resistance system of T. thermophilus in more detail. We employed TtSmtB-based pull-down assays with protein extracts from cultures treated with arsenate and arsenite to obtain an S-adenosylmethionine (SAM)-dependent arsenite methyltransferase (TtArsM). In vivo and in vitro analyses were performed to shed light on this new component of the arsenic resistance network and its peculiar catalytic mechanism. Heterologous expression of TtarsM in Escherichia coli resulted in arsenite detoxification at mesophilic temperatures. Although TtArsM does not contain a canonical arsenite binding site, the purified protein does catalyse SAM-dependent arsenite methylation. In addition, in vitro analyses confirmed the unique interaction between TtArsM and TtSmtB. Next, a highly efficient ThermoCas9-based genome-editing tool was developed to delete the TtArsM-encoding gene on the T. thermophilus genome, and to confirm its involvement in the arsenite detoxification system. Finally, the TtarsX efflux pump gene in the T. thermophilus ΔTtarsM genome was substituted by a gene, encoding a stabilised yellow fluorescent protein (sYFP), to create a sensitive genome-based bioreporter system for the detection of arsenic ions.

FGF-7 modulates ureteric bud growth and nephron number in the developing kidney

Development ◽  
1999 ◽  
Vol 126 (3) ◽  
pp. 547-554 ◽  
Author(s):  
J. Qiao ◽  
R. Uzzo ◽  
T. Obara-Ishihara ◽  
L. Degenstein ◽  
E. Fuchs ◽  
...  
Keyword(s):  
Expression Patterns ◽  
Body Volume ◽  
Metanephric Mesenchyme ◽  
Nephron Number ◽  
Wild Type ◽  
Ureteric Bud ◽  
Kidney Size ◽  
Bud Growth ◽  
Collecting System

The importance of proportioning kidney size to body volume was established by clinical studies which demonstrated that in-born defecits of nephron number predispose the kidney to disease. As the kidney develops, the expanding ureteric bud or renal collecting system induces surrounding metanephric mesenchyme to proliferate and differentiate into nephrons. Thus, it is likely that nephron number is related to ureteric bud growth. The expression patterns of mRNAs encoding Fibroblast Growth Factor-7 (FGF-7) and its high affinity receptor suggested that FGF-7 signaling may play a role in regulating ureteric bud growth. To test this hypothesis we examined kidneys from FGF-7-null and wild-type mice. Results of these studies demonstrate that the developing ureteric bud and mature collecting system of FGF-7-null kidneys is markedly smaller than wild type. Furthermore, morphometric analyses indicate that mature FGF-7-null kidneys have 30+/−6% fewer nephrons than wild-type kidneys. In vitro experiments demonstrate that elevated levels of FGF-7 augment ureteric bud growth and increase the number of nephrons that form in rodent metanephric kidney organ cultures. Collectively, these results demonstrate that FGF-7 levels modulate the extent of ureteric bud growth during development and the number of nephrons that eventually form in the kidney.

scholarly journals The Maize Clade A PP2C Phosphatases Play Critical Roles in Multiple Abiotic Stress Responses

2019 ◽  
Vol 20 (14) ◽  
pp. 3573 ◽  
Author(s):  
Zhenghua He ◽  
Jinfeng Wu ◽  
Xiaopeng Sun ◽  
Mingqiu Dai
Keyword(s):  
Drought Tolerance ◽  
Stress Responses ◽  
Transgenic Arabidopsis ◽  
Expression Patterns ◽  
Survival Rates ◽  
Wild Type ◽  
Multiple Stresses ◽  
Core Components ◽  
Leaf Water Loss ◽  
Natural Variations

As the core components of abscisic acid (ABA) signal pathway, Clade A PP2C (PP2C-A) phosphatases in ABA-dependent stress responses have been well studied in Arabidopsis. However, the roles and natural variations of maize PP2C-A in stress responses remain largely unknown. In this study, we investigated the expression patterns of ZmPP2C-As treated with multiple stresses and generated transgenic Arabidopsis plants overexpressing most of the ZmPP2C-A genes. The results showed that the expression of most ZmPP2C-As were dramatically induced by multiple stresses (drought, salt, and ABA), indicating that these genes may have important roles in response to these stresses. Compared with wild-type plants, ZmPP2C-A1, ZmPP2C-A2, and ZmPP2C-A6 overexpression plants had higher germination rates after ABA and NaCl treatments. ZmPP2C-A2 and ZmPP2C-A6 negatively regulated drought responses as the plants overexpressing these genes had lower survival rates, higher leaf water loss rates, and lower proline accumulation compared to wild type plants. The natural variations of ZmPP2C-As associated with drought tolerance were also analyzed and favorable alleles were detected. We widely studied the roles of ZmPP2C-A genes in stress responses and the natural variations detected in these genes have the potential to be used as molecular markers in genetic improvement of maize drought tolerance.

scholarly journals The Receptor for Parathyroid Hormone and Parathyroid Hormone-Related Peptide Is Hydrolyzed and Its Signaling Properties Are Altered by Directly Binding the Calpain Small Subunit

Endocrinology ◽  
2005 ◽  
Vol 146 (5) ◽  
pp. 2336-2344 ◽  
Author(s):  
Masako Shimada ◽  
Matthew J. Mahon ◽  
Peter A. Greer ◽  
Gino V. Segre
Keyword(s):  
Parathyroid Hormone ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Small Subunit ◽  
Hek293 Cells ◽  
Calpain Inhibitor ◽  
Dependent Manner ◽  
Intact Cells ◽  
Calcium Dependent

Abstract We show calcium-dependent, direct binding between the N-terminal portion of the PTH/PTHrP receptor (PTH1R) C-terminal intracellular tail and the calpain small subunit. Binding requires, but may not be limited to, amino acids W474, S475, and W477. The wild-type, full-length rat (r) PTH1R, but not rPTH1R with W474A/W477A substitutions, copurifies with the endogenous calpain small subunit in HEK293 cells. Calpain hydrolyzes ΔNt-rPTH1R, a receptor with a 156-amino acid N-terminal deletion, in a calcium-dependent manner in vitro and in intact cells. Most importantly, PTH stimulation increases the cleavage of ΔNt-rPTH1R and rPTH1R-yellow fluorescent protein in HEK293 cells, and of talin in HEK293 cells expressing rPTH1R-yellow fluorescent protein and in ROS17/2.8 osteoblast-like cells that express rPTH1R endogenously. The absence of calpain in Capn4-null embryonic fibroblasts and the lowered calpain activity in MC3T3-E1 osteoblastic cells due to stable expression of the calpain inhibitor, calpastatin, reduce PTH-stimulated cAMP accumulation. The calpain small subunit is the second protein, in addition to the sodium-hydrogen exchanger regulatory factor, and the first enzyme that binds the PTH1R; PTH1R bound to both of these proteins results in altered PTH signaling.

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