TWIK-1 BAC-GFP Transgenic Mice, an Animal Model for TWIK-1 Expression

TWIK-1 is the first identified member of the two-pore domain potassium (K2P) channels that are involved in neuronal excitability and astrocytic passive conductance in the brain. Despite the physiological roles of TWIK-1, there is still a lack of information on the basic expression patterns of TWIK-1 proteins in the brain. Here, using a modified bacterial artificial chromosome (BAC), we generated a transgenic mouse (Tg mouse) line expressing green fluorescent protein (GFP) under the control of the TWIK-1 promoter (TWIK-1 BAC-GFP Tg mice). We confirmed that nearly all GFP-producing cells co-expressed endogenous TWIK-1 in the brain of TWIK-1 BAC-GFP Tg mice. GFP signals were highly expressed in various brain areas, including the dentate gyrus (DG), lateral entorhinal cortex (LEC), and cerebellum (Cb). In addition, we found that GFP signals were highly expressed in immature granule cells in the DG. Finally, our TWIK-1 BAC-GFP Tg mice mimic the upregulation of TWIK-1 mRNA expression in the hippocampus following the injection of kainic acid (KA). Our data clearly showed that TWIK-1 BAC-GFP Tg mice are a useful animal model for studying the mechanisms regulating TWIK-1 gene expression and the physiological roles of TWIK-1 channels in the brain.

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Transgenic Mice Expressing Green Fluorescent Protein under the Control of the Corticotropin-Releasing Hormone Promoter

Endocrinology ◽

10.1210/en.2009-0881 ◽

2009 ◽

Vol 150 (12) ◽

pp. 5626-5632 ◽

Cited By ~ 38

Author(s):

Tamar Alon ◽

Ligang Zhou ◽

Cristian A. Pérez ◽

Alastair S. Garfield ◽

Jeffrey M. Friedman ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Fluorescent Protein ◽

Artificial Chromosome ◽

Brain Regions ◽

Ingestive Behavior ◽

Physiological Processes ◽

Functional Relevance ◽

Green Fluorescent ◽

And Function ◽

The Brain

Abstract CRH is widely expressed in the brain and is of broad functional relevance to a number of physiological processes, including stress response, parturition, immune response, and ingestive behavior. To delineate further the organization of the central CRH network, we generated mice expressing green fluorescent protein (GFP) under the control of the CRH promoter, using bacterial artificial chromosome technology. Here we validate CRH-GFP transgene expression within specific brain regions and confirm the distribution of central GFP-producing cells to faithfully recapitulate that of CRH-expressing cells. Furthermore, we confirm the functional integrity of a population of GFP-producing cells by demonstrating their apposite responsiveness to nutritional status. We anticipate that this transgenic model will lend itself as a highly tractable tool for the investigation of CRH expression and function in discrete brain regions.

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Systemic AAV6-synapsin-GFP administration results in lower liver biodistribution, compared to AAV1&2 and AAV9, with neuronal expression following ultrasound-mediated brain delivery

Scientific Reports ◽

10.1038/s41598-021-81046-5 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Danielle Weber-Adrian ◽

Rikke Hahn Kofoed ◽

Joseph Silburt ◽

Zeinab Noroozian ◽

Kairavi Shah ◽

...

Keyword(s):

Gene Delivery ◽

Intravenous Injection ◽

Viral Vectors ◽

Focused Ultrasound ◽

Fluorescent Protein ◽

Gfp Expression ◽

Peripheral Organs ◽

Neuronal Expression ◽

Green Fluorescent ◽

The Brain

AbstractNon-surgical gene delivery to the brain can be achieved following intravenous injection of viral vectors coupled with transcranial MRI-guided focused ultrasound (MRIgFUS) to temporarily and locally permeabilize the blood–brain barrier. Vector and promoter selection can provide neuronal expression in the brain, while limiting biodistribution and expression in peripheral organs. To date, the biodistribution of adeno-associated viruses (AAVs) within peripheral organs had not been quantified following intravenous injection and MRIgFUS delivery to the brain. We evaluated the quantity of viral DNA from the serotypes AAV9, AAV6, and a mosaic AAV1&2, expressing green fluorescent protein (GFP) under the neuron-specific synapsin promoter (syn). AAVs were administered intravenously during MRIgFUS targeting to the striatum and hippocampus in mice. The syn promoter led to undetectable levels of GFP expression in peripheral organs. In the liver, the biodistribution of AAV9 and AAV1&2 was 12.9- and 4.4-fold higher, respectively, compared to AAV6. The percentage of GFP-positive neurons in the FUS-targeted areas of the brain was comparable for AAV6-syn-GFP and AAV1&2-syn-GFP. In summary, MRIgFUS-mediated gene delivery with AAV6-syn-GFP had lower off-target biodistribution in the liver compared to AAV9 and AAV1&2, while providing neuronal GFP expression in the striatum and hippocampus.

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Green fluorescent protein (GFP) expression patterns in the olfactory epithelium of GFP transgenic cloned Jinhua pigs

Acta Zoologica ◽

10.1111/azo.12029 ◽

2013 ◽

Vol 95 (3) ◽

pp. 319-329

Author(s):

Atsushi Hirao ◽

Tatsuo Kawarasaki ◽

Kenjiro Konno ◽

Satoko Enya ◽

Masatoshi Shibata ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Olfactory Epithelium ◽

Fluorescent Protein ◽

Expression Patterns ◽

Gfp Expression ◽

Green Fluorescent

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GATA-1 expression pattern can be recapitulated in living transgenic zebrafish using GFP reporter gene

Development ◽

10.1242/dev.124.20.4105 ◽

1997 ◽

Vol 124 (20) ◽

pp. 4105-4111 ◽

Cited By ~ 6

Author(s):

Q. Long ◽

A. Meng ◽

H. Wang ◽

J.R. Jessen ◽

M.J. Farrell ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Progenitor Cells ◽

Reporter Gene ◽

Fluorescent Protein ◽

Expression Patterns ◽

Transgenic Fish ◽

Green Fluorescent Protein Reporter ◽

Erythroid Progenitor ◽

Green Fluorescent ◽

Fluorescent Protein Reporter

In this study, DNA constructs containing the putative zebrafish promoter sequences of GATA-1, an erythroid-specific transcription factor, and the green fluorescent protein reporter gene, were microinjected into single-cell zebrafish embryos. Erythroid-specific activity of the GATA-1 promoter was observed in living embryos during early development. Fluorescent circulating blood cells were detected in microinjected embryos 24 hours after fertilization and were still present in 2-month-old fish. Germline transgenic fish obtained from the injected founders continued to express green fluorescent protein in erythroid cells in the F1 and F2 generations. The green fluorescent protein expression patterns in transgenic fish were consistent with the pattern of GATA-1 mRNA expression detected by RNA in situ hybridization. These transgenic fish have allowed us to isolate, by fluorescence-activated cell sorting, the earliest erythroid progenitor cells from developing embryos for in vitro studies. By generating transgenic fish using constructs containing other zebrafish promoters and green fluorescent protein reporter gene, it should be possible to visualize the origin and migration of any lineage-specific progenitor cells in a living embryo.

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Quorum-Sensing Genes in Pseudomonas aeruginosa Biofilms: Their Role and Expression Patterns

Applied and Environmental Microbiology ◽

10.1128/aem.67.4.1865-1873.2001 ◽

2001 ◽

Vol 67 (4) ◽

pp. 1865-1873 ◽

Cited By ~ 341

Author(s):

Teresa R. De Kievit ◽

Richard Gillis ◽

Steve Marx ◽

Chris Brown ◽

Barbara H. Iglewski

Keyword(s):

Pseudomonas Aeruginosa ◽

Quorum Sensing ◽

Biofilm Formation ◽

Fluorescent Protein ◽

Expression Patterns ◽

Homoserine Lactone ◽

Lower Percentage ◽

Spatial Studies ◽

Green Fluorescent ◽

Biofilm Development

ABSTRACT Acylated homoserine lactone molecules are used by a number of gram-negative bacteria to regulate cell density-dependent gene expression by a mechanism known as quorum sensing (QS). InPseudomonas aeruginosa, QS or cell-to-cell signaling controls expression of a number of virulence factors, as well as biofilm differentiation. In this study, we investigated the role played by the las and rhl QS systems during the early stages of static biofilm formation when cells are adhering to a surface and forming microcolonies. These studies revealed a marked difference in biofilm formation between the PAO1 parent and the QS mutants when glucose, but not citrate, was used as the sole carbon source. To further elucidate the contribution of lasI andrhlI to biofilm maturation, we utilized fusions to unstable green fluorescent protein in concert with confocal microscopy to perform real-time temporal and spatial studies of these genes in a flowing environment. During the course of 8-day biofilm development,lasI expression was found to progressively decrease over time. Conversely, rhlI expression remained steady throughout biofilm development but occurred in a lower percentage of cells. Spatial analysis revealed that lasI andrhlI were maximally expressed in cells located at the substratum and that expression decreased with increasing biofilm height. Because QS was shown previously to be involved in biofilm differentiation, these findings have important implications for the design of biofilm prevention and eradication strategies.

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In vivo analysis of key elements within the renin regulatory region

Physiological Genomics ◽

10.1152/physiolgenomics.00017.2008 ◽

2008 ◽

Vol 35 (3) ◽

pp. 243-253 ◽

Cited By ~ 22

Author(s):

Sean T. Glenn ◽

Craig A. Jones ◽

Li Pan ◽

Kenneth W. Gross

Keyword(s):

Fluorescent Protein ◽

Regulatory Region ◽

Renin Angiotensin System ◽

Artificial Chromosome ◽

Proximal Promoter ◽

Gfp Expression ◽

Renin Gene ◽

Green Fluorescent

Renin is responsible for initiating the enzymatic cascade that results in the production of angiotensin II, the major effector molecule of the renin-angiotensin system (RAS). Extensive information on the regulatory region of the renin gene has been derived by transient transfection studies in vitro, particularly using the As4.1 cell line. To verify key factors within the regulatory region of renin in vivo, homologous recombination was used to introduce a green fluorescent protein (GFP) cassette into exon one of the renin gene contained within a 240 kb bacterial artificial chromosome (BAC) to create a construct that has GFP expression controlled by the renin regulatory region (RenGFP BAC). Within the regulatory region of the RenGFP BAC construct we independently deleted the enhancer, as well as mutated the HOX-PBX site within the proximal promoter element. Transgenic lines were generated for each of these BAC constructs and GFP expression was analyzed throughout a spectrum of tissues positive for renin expression including the kidney, adrenal gland, gonadal artery, and submandibular gland. The results described within this manuscript support the interpretation that the renin enhancer is critical for regulating baseline expression where as the Hox/Pbx site is important for the tissue specificity of renin expression.

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Nuclear localization of angiotensinogen in astrocytes

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00594.2004 ◽

2005 ◽

Vol 288 (2) ◽

pp. R539-R546 ◽

Cited By ~ 48

Author(s):

Mikhiela Sherrod ◽

Xuebo Liu ◽

Xiaoji Zhang ◽

Curt D. Sigmund

Keyword(s):

Fusion Protein ◽

Nuclear Localization ◽

Fluorescent Protein ◽

Primary Cultures ◽

Endocrine Function ◽

Astrocytoma Cell ◽

Protein Encoding ◽

Localization Signal ◽

Green Fluorescent ◽

The Brain

In the brain, angiotensinogen (AGT) is primarily expressed in astrocytes; brain ANG II derived from locally produced AGT has been shown to influence blood pressure. To better understand the molecular basis of AGT expression in the brain, we identified a human astrocytoma cell line, CCF-STTG1, that expresses endogenous AGT mRNA and produces AGT protein. Studies examining CCF-STTG1 cell AGT after N- and O-glycosidase suggest that AGT may not be posttranslationally modified by glycosylation in these cells as it is in plasma. Small amounts of AGT (5% of HepG2) were detected in the culture medium, suggesting a low rate of AGT secretion. Immunocytochemical examination of AGT in CCF-STTG1 cells revealed mainly nuclear localization. Although this has not been previously reported, it is consistent with nuclear localization of other serpin family members. To examine this further, we generated a fusion protein consisting of green fluorescent protein (GFP) and human AGT and examined subcellular localization by confocal microscopy after confirming expression of the fusion protein by Western blot. In CCF-STTG1 cells, a control GFP construct lacking AGT was mainly localized in the cytoplasm, whereas the GFP-AGT fusion protein was primarily localized in the nucleus. To map the location of a potential nuclear localization signal, overlapping 500-bp fragments of human AGT cDNA were fused in frame downstream of GFP. Although four of the fusion proteins exhibited either perinuclear or cytoplasmic localization, one fusion protein encoding the COOH terminus of AGT was localized in the nucleus. Importantly, nuclear localization of human AGT was confirmed in primary cultures of glial cells isolated from transgenic mice expressing the human AGT under the control of its own endogenous promoter. Our results suggest that AGT may have a novel intracellular role in the brain apart from its predicted endocrine function.

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Iba1+/NG2+ Macrophage-Like Cells Expressing a Variety of Neuroprotective Factors Ameliorate Ischemic Damage of the Brain

Journal of Cerebral Blood Flow & Metabolism ◽

10.1038/jcbfm.2009.233 ◽

2009 ◽

Vol 30 (3) ◽

pp. 603-615 ◽

Cited By ~ 51

Author(s):

Anna Smirkin ◽

Hiroaki Matsumoto ◽

Hisaaki Takahashi ◽

Akihiro Inoue ◽

Masahiko Tagawa ◽

...

Keyword(s):

Bone Marrow ◽

Fluorescent Protein ◽

Artery Occlusion ◽

Brain Lesions ◽

Ischemic Lesion ◽

Ischemic Brain ◽

Green Fluorescent ◽

High Level ◽

Cerebral Artery Occlusion ◽

The Brain

In a transient 90-min middle cerebral artery occlusion (MCAO) model of rats, a large ischemic lesion is formed where macrophage-like cells massively accumulate, many of which express a macrophage marker, Iba1, and an oligodendrocyte progenitor cell marker, NG2 chondroitin sulfate proteoglycan (NG2); therefore, the cells were termed BINCs (Brain Iba1+/NG2+Cells). A bone marrow transplantation experiment using green-fluorescent protein-transgenic rats showed that BINCs were derived from bone marrow. 5-Fluorouracil (5FU) injection at 2 days post reperfusion (2 dpr) markedly reduced the number of BINCs at 7 dpr, causing enlargement of necrotic volumes and frequent death of the rats. When isolated BINCs were transplanted into 5FU-aggravated ischemic lesion, the volume of the lesion was much reduced. Quantitative real-time RT-PCR showed that BINCs expressed mRNAs encoding bFGF, BMP2, BMP4, BMP7, GDNF, HGF, IGF-1, PDGF-A, and VEGF. In particular, BINCs expressed IGF-1 mRNA at a very high level. Immunohistochemical staining showed that IGF-1-expressing BINCs were found not only in rat but also human ischemic brain lesions. These results suggest that bone marrow-derived BINCs play a beneficial role in ischemic brain lesions, at least in part, through secretion of neuroprotective factors.

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In vivo analysis of mouse gastrin gene regulation in enhanced GFP-BAC transgenic mice

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00134.2010 ◽

2011 ◽

Vol 300 (2) ◽

pp. G334-G344 ◽

Cited By ~ 19

Author(s):

Shigeo Takaishi ◽

Wataru Shibata ◽

Hiroyuki Tomita ◽

Guangchun Jin ◽

Xiangdong Yang ◽

...

Keyword(s):

Transcriptional Control ◽

Fluorescent Protein ◽

Artificial Chromosome ◽

Transitional Zone ◽

Anterior Lobe ◽

Gastric Antrum ◽

Neuroendocrine Cells ◽

Adult Mice ◽

Green Fluorescent

Gastrin is secreted from a subset of neuroendocrine cells residing in the gastric antrum known as G cells, but low levels are also expressed in fetal pancreas and intestine and in many solid malignancies. Although past studies have suggested that antral gastrin is transcriptionally regulated by inflammation, gastric pH, somatostatin, and neoplastic transformation, the transcriptional regulation of gastrin has not previously been demonstrated in vivo. Here, we describe the creation of an enhanced green fluorescent protein reporter (mGAS-EGFP) mouse using a bacterial artificial chromosome that contains the entire mouse gastrin gene. Three founder lines expressed GFP signals in the gastric antrum and the transitional zone to the corpus. In addition, GFP(+) cells could be detected in the fetal pancreatic islets and small intestinal villi, but not in these organs of the adult mice. The administration of acid-suppressive reagents such as proton pump inhibitor omeprazole and gastrin/CCK-2 receptor antagonist YF476 significantly increased GFP signal intensity and GFP(+) cell numbers in the antrum, whereas these parameters were decreased by overnight fasting, octreotide (long-lasting somatostatin ortholog) infusion, and Helicobacter felis infection. GFP(+) cells were also detected in the anterior lobe of the pituitary gland and importantly in the colonic tumor cells induced by administration with azoxymethane and dextran sulfate sodium salt. This transgenic mouse provides a useful tool to study the regulation of mouse gastrin gene in vivo, thus contributing to our understanding of the mechanisms involved in transcriptional control of the gastrin gene.

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