Live imaging of apoptosis in a novel transgenic mouse highlights its role in neural tube closure

Yamaguchi, Yoshifumi; Shinotsuka, Naomi; Nonomura, Kazuteru; Takemoto, Kiwamu; Kuida, Keisuke; Yosida, Hiroki; Miura, Masayuki

doi:10.1083/jcb.201104057

Cited by 167 publications

(183 citation statements)

References 64 publications

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“…In many studies that reported the generation of transgenic mice expressing FRET biosensors, analysis was conducted in vitro with cells isolated from the tissues of transgenic mice (Atkin et al, 2009;Hara et al, 2004;Isotani et al, 2004;Nikolaev et al, 2006;Tsujino et al, 2005), and only a few of them succeeded in FRET imaging in living tissues (Atkin et al, 2009;Tsujino et al, 2005), probably due to low levels of expression. Recently, Yamaguchi et al succeeded in the generation of transgenic mice expressing a FRET biosensor for Caspase3 by introducing insulator sequences (Yamaguchi et al, 2011). The authors suggested that the low expression of FRET biosensors is caused by silencing of the transgene.…”

Section: Discussionmentioning

confidence: 99%

Live Imaging of Protein Kinase Activities in Transgenic Mice Expressing FRET Biosensors

Kamioka

Sumiyama

Mizuno

et al. 2012

Cell Struct. Funct.

130

155

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ABSTRACT. Genetically-encoded biosensors based on the principle of Förster resonance energy transfer (FRET) have been widely used in biology to visualize the spatiotemporal dynamics of signaling molecules. Despite the increasing multitude of these biosensors, their application has been mostly limited to cultured cells with transient biosensor expression, due to particular difficulties in the development of transgenic mice that express FRET biosensors. In this study, we report the efficient generation of transgenic mouse lines expressing heritable and functional biosensors for ERK and PKA. These transgenic mice were created by the cytoplasmic co-injection of Tol2 transposase mRNA and a circular plasmid harbouring Tol2 recombination sites. High expression of the biosensors in a wide range of cell types allowed us to screen newborn mice simply by inspection. Observation of these transgenic mice by two-photon excitation microscopy yielded real-time activity maps of ERK and PKA in various tissues, with greatly improved signal-to-background ratios. Our transgenic mice may be bred into diverse genetic backgrounds; moreover, the protocol we have developed paves the way for the generation of transgenic mice that express other FRET biosensors, with important applications in the characterization of physiological and pathological signal transduction events in addition to drug development and screening.

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Section: Discussionmentioning

confidence: 99%

Live Imaging of Protein Kinase Activities in Transgenic Mice Expressing FRET Biosensors

Kamioka

Sumiyama

Mizuno

et al. 2012

Cell Struct. Funct.

130

155

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“…Live imaging in transgenic mouse model expressing SCAT3 revealed well-coordinated apoptosis cell death during the process of neural tube closure (31).…”

Section: Adipocyte Death Macrophage Activationmentioning

confidence: 99%

Adipocyte Death and Chronic Inflammation in Obesity

Kuroda

Sakaue

2017

J. Med. Invest.

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: Cell death is closely linked to many diseases including cancer, neurodegenerative diseases, autoimmune diseases, and metabolic disorders. Increased adipocyte death has been reported during the development of obesity. Adipocyte death may be caused by excessive stress during obesity-related adipose tissue remodeling. Adipose tissue macrophages are key players in obesity-related inflammation and systemic insulin resistance. Accumulating evidence suggests that adipocyte death is involved in immune cell function and initiates inflammation through an interaction with macrophages ; however, the precise mechanisms remain largely unknown.This review focuses on the contribution of dead cells (particularly dead adipocytes in adipose tissue) to the pathophysiological conditions associated with obesity. J. Med. Invest.

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“…When these mice are crossed with lineage-specific Cre mice, the cells with Cre recombinase begin to express FRET biosensors. Finally, another group succeeded in generating a transgenic mouse expressing SCAT3, a single-molecule FRET biosensor that reports Caspase-3 activity, by a conventional, linearized DNA injection method but using chicken HS4 insulator sequences to overcome silencing of the transgene (20).…”

Section: Generation Of Stable Cell Lines and Transgenic Mice Expressimentioning

confidence: 99%

“…Because of their high signal/noise ratio and wider application in live imaging, and because their use does not require calculation of the corrective FRET efficiency, many laboratories have preferentially created single-molecule FRET biosensors that monitor specific molecules such as kinases (6)(7)(8)(9)(10), proteases (11)(12)(13), GTPases (14)(15)(16)(17) or lipids (18). Recently, a breakthrough was reported in the generation of transgenic mice expressing single-molecular FRET biosensors, in which the activities of numerous kinases, proteases, or GTPases are visualized in living tissues and organs (19)(20)(21). Despite the remarkable technical expansion of FRET microscopy, a couple of problems in image acquisition and data analysis have hindered precise signal quantification in intravital application.…”

mentioning

confidence: 99%

Future Perspective of Single-Molecule FRET Biosensors and Intravital FRET Microscopy

Hirata

Kiyokawa

2016

Biophysical Journal

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Fö rster (or fluorescence) resonance energy transfer (FRET) is a nonradiative energy transfer process between two fluorophores located in close proximity to each other. To date, a variety of biosensors based on the principle of FRET have been developed to monitor the activity of kinases, proteases, GTPases or lipid concentration in living cells. In addition, generation of biosensors that can monitor physical stresses such as mechanical power, heat, or electric/magnetic fields is also expected based on recent discoveries on the effects of these stressors on cell behavior. These biosensors can now be stably expressed in cells and mice by transposon technologies. In addition, two-photon excitation microscopy can be used to detect the activities or concentrations of bioactive molecules in vivo. In the future, more sophisticated techniques for image acquisition and quantitative analysis will be needed to obtain more precise FRET signals in spatiotemporal dimensions. Improvement of tissue/organ position fixation methods for mouse imaging is the first step toward effective image acquisition. Progress in the development of fluorescent proteins that can be excited with longer wavelength should be applied to FRET biosensors to obtain deeper structures. The development of computational programs that can separately quantify signals from single cells embedded in complicated three-dimensional environments is also expected. Along with the progress in these methodologies, two-photon excitation intravital FRET microscopy will be a powerful and valuable tool for the comprehensive understanding of biomedical phenomena.Since the discovery of green fluorescent protein (GFP) (1), scientists have been widely employing this gene-encoded fluorescent protein (FP) to investigate the spatiotemporal dynamics of molecules with subcellular resolution. One of the applications of FP engineering is the development of biosensors based on the principle of Förster (or fluorescence) resonance energy transfer (FRET) (2). FRET is a nonradiative energy transfer process between two fluorophores located in close proximity to each other, and its efficiency is strongly dependent on the distance between the fluorophores. More precisely, the energy transfer rate (k t ) from a donor to an acceptor fluorophore is calculated in the following Förster's equation;where J is the overlap of donor emission and acceptor excitation spectra, k 2 is an orientation factor of transition moment (a factor determined by the relative orientation of the two fluorophores), n is a refractive index, R is the distance between the donor and acceptor fluorophores, and k f is the donor fluorescence emission speed. Therefore, FRET efficiency is determined by the distance and orientation of the two fluorophores. To measure FRET efficiency, there are roughly two methods; ratiometric analysis and lifetime analysis. Ratiometric analysis utilizes fluorescence intensity of an acceptor (e.g., yellow FP (YFP)) and a donor (e.g., cyan FP (CFP)) upon the donor excitation. Because accept...

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Live imaging of apoptosis in a novel transgenic mouse highlights its role in neural tube closure

Abstract: Imaging of caspase activation in living mouse embryos during development suggests that caspase-mediated cell removal facilitates neural tube closure in a temporally regulated manner.

Cited by 167 publications

References 64 publications

Live Imaging of Protein Kinase Activities in Transgenic Mice Expressing FRET Biosensors

Live Imaging of Protein Kinase Activities in Transgenic Mice Expressing FRET Biosensors

Adipocyte Death and Chronic Inflammation in Obesity

Future Perspective of Single-Molecule FRET Biosensors and Intravital FRET Microscopy

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