Non-invasive visualization of the lipid product of class I PI3K in transgenic mouse models

2007 ◽  
Vol 35 (2) ◽  
pp. 215-218 ◽  
Author(s):  
T. Sasaki ◽  
J. Sasaki ◽  
K. Watanabe ◽  
A. Suzuki
Keyword(s):  
Transgenic Mice ◽  
Fluorescent Protein ◽  
Neutrophil Migration ◽  
Pleckstrin Homology Domain ◽  
Inositol Phosphatase ◽  
Lipid Product ◽  
Src Homology ◽  
Spatio Temporal ◽  
Metabolizing Enzyme ◽  
Green Fluorescent

PI3Ks (phosphoinositide 3-kinases) regulate many critical cellular responses by producing PI(3,4,5)P3 (phosphatidylinositol 3,4,5-trisphosphate). To facilitate the spatio-temporal characterization of PI(3,4,5)P3 in living primary cells, we generated a novel strain of transgenic mice [AktPH (Akt pleckstrin homology domain)–GFP (green fluorescent protein) Tg (transgenic) mice] that express a fluorescent bioprobe for PI(3,4,5)P3/PI(3,4)P2 (phosphatidylinositol 3,4-bisphosphate). By crossing AktPH–GFP Tg mice with strains of gene-targeted ‘knockout’ mice lacking a particular phosphoinositide-metabolizing enzyme, we have been able to evaluate the contribution of each enzyme to PI(3,4,5)P3 localization in migrating neutrophils. Our results indicate that PI3Kγ and the PI(3,4,5)P3 phosphatase SHIP1 [SH2 (Src homology 2)-containing inositol phosphatase-1] are the key regulators of PI(3,4,5)P3 dynamics during fMet-Leu-Phe (N-formylmethionyl-leucylphenylalanine; ‘chemotactic peptide’)-stimulated neutrophil migration. Our study has also validated the fluorescent transgenic strategy for studying PI(3,4,5)P3 metabolism in physiological and pathological situations.

scholarly journals Inhibitory receptor FcγRIIb mediates the effects of IgG on a phagosome acidification and a sequential dephosphorylation system comprising SHIPs and Inpp4a

2017 ◽  
Vol 23 (4) ◽  
pp. 401-409 ◽  
Author(s):  
Tomohiro Segawa ◽  
Kaoru Hazeki ◽  
Kiyomi Nigorikawa ◽  
Atsuko Nukuda ◽  
Tomoki Tanizawa ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Fluorescence Probe ◽  
Pleckstrin Homology Domain ◽  
Raw 264.7 Macrophages ◽  
Lipid Phosphatases ◽  
Src Homology 2 ◽  
Lipid Phosphatase ◽  
Src Homology ◽  
Src Homology 2 Domain ◽  
Green Fluorescent

The relative abundance of phosphoinositide (PI) species on the phagosome membrane fluctuates over the course of phagocytosis. PtdIns(3,4,5)P3 and PtdIns(3,4)P2 rapidly increase in the forming of the phagocytic cup, following which they disappear after sealing of the cup. In the present study, we monitored the clearance of these PI species using the enhanced green fluorescent protein-fused pleckstrin homology domain of Akt, a fluorescence probe that binds both PtdIns(3,4,5)P3 and PtdIns(3,4)P2 in Raw 264.7 macrophages. The clearance of PIs was much faster when the phagocytosed particles were coated with IgG. The effect of IgG was not observed in the macrophages deficient in FcγRIIb, an inhibitory IgG receptor. To identify the lipid phosphatases responsible for the FcγRIIb-accelerated PI clearance, we prepared a panel of lipid phosphatase-deficient cells. The lack of a PI 5-phosphatase Src homology 2 domain-containing inositol-5-phosphatase (SHIP)1 or SHIP2 impaired the FcγRIIb-accelerated clearance of PIs. The lack of a PI 4-phosphatase Inpp4a also impaired the accelerated PIs clearance. In the FcγRIIb- and Inpp4a-deficient cells, acidification of the formed phagosome was slowed. These results suggested that FcγRIIb drives the sequential dephosphorylation system comprising SHIPs and Inpp4a, and accelerates phagosome acidification.

High level Purkinje cell specific expression of green fluorescent protein in transgenic mice

2001 ◽  
Vol 115 (6) ◽  
pp. 455-464 ◽  
Author(s):  
Xulun Zhang ◽  
Stephan L. Baader ◽  
Feng Bian ◽  
Wolfgang Müller ◽  
John Oberdick
Keyword(s):  
Green Fluorescent Protein ◽  
Transgenic Mice ◽  
Purkinje Cell ◽  
Fluorescent Protein ◽  
Specific Expression ◽  
Green Fluorescent ◽  
High Level

BAC transgenic mice express enhanced green fluorescent protein in central and peripheral cholinergic neurons

2006 ◽  
Vol 27 (3) ◽  
pp. 391-397 ◽  
Author(s):  
Yvonne N. Tallini ◽  
Bo Shui ◽  
Kai Su Greene ◽  
Ke-Yu Deng ◽  
Robert Doran ◽  
...  
Keyword(s):  
Nervous System ◽  
Green Fluorescent Protein ◽  
Transgenic Mice ◽  
Peripheral Nervous System ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Cholinergic Neurons ◽  
Autonomic Nerves ◽  
Organ Systems ◽  
Green Fluorescent

The peripheral nervous system has complex and intricate ramifications throughout many target organ systems. To date this system has not been effectively labeled by genetic markers, due largely to inadequate transcriptional specification by minimum promoter constructs. Here we describe transgenic mice in which enhanced green fluorescent protein (eGFP) is expressed under the control of endogenous choline acetyltransferase (ChAT) transcriptional regulatory elements, by knock-in of eGFP within a bacterial artificial chromosome (BAC) spanning the ChAT locus and expression of this construct as a transgene. eGFP is expressed in ChATBAC-eGFP mice in central and peripheral cholinergic neurons, including cell bodies and processes of the somatic motor, somatic sensory, and parasympathetic nervous system in gastrointestinal, respiratory, urogenital, cardiovascular, and other peripheral organ systems. Individual epithelial cells and a subset of lymphocytes within the gastrointestinal and airway mucosa are also labeled, indicating genetic evidence of acetylcholine biosynthesis. Central and peripheral neurons were observed as early as 10.5 days postcoitus in the developing mouse embryo. ChATBAC-eGFP mice allow excellent visualization of all cholinergic elements of the peripheral nervous system, including the submucosal enteric plexus, preganglionic autonomic nerves, and skeletal, cardiac, and smooth muscle neuromuscular junctions. These mice should be useful for in vivo studies of cholinergic neurotransmission and neuromuscular coupling. Moreover, this genetic strategy allows the selective expression and conditional inactivation of genes of interest in cholinergic nerves of the central nervous system and peripheral nervous system.

Green fluorescent protein-transgenic mice: immune functions and their application to studies of lymphocyte development

2000 ◽  
Vol 70 (3) ◽  
pp. 165-171 ◽  
Author(s):  
Naoto Kawakami ◽  
Naoki Sakane ◽  
Fumiko Nishizawa ◽  
Mutsumi Iwao ◽  
So-ichiro Fukada ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Transgenic Mice ◽  
Fluorescent Protein ◽  
Lymphocyte Development ◽  
Immune Functions ◽  
Green Fluorescent
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