Impact of embryonic expression of enhanced green fluorescent protein on early mouse development

Devgan, Vikram; M, Rao; Seshagiri, Polani B.

doi:10.1016/j.bbrc.2003.11.184

Cited by 37 publications

(27 citation statements)

References 18 publications

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“…Cellular stress responses have also been reported in cultured cells stably expressing GFP (Zhang et al, 2003), and eGFP expression has also been reported to increase production of superoxide and hydrogen peroxide (Ganini et al, 2017). Adverse effects have also been observed in transgenic GFP animals (Devgan et al, 2004;Huang et al, 2000;Mawhinney and Staveley, 2011). Therefore, despite GFP tagging of proteins driving huge advances in our understanding of biological processes, GFP is not as inert as previously assumed.…”

Section: Introductionmentioning

confidence: 99%

An improved Akt reporter reveals intra- and inter-cellular heterogeneity and oscillations in signal transduction

Norris

Yang

Krycer

et al. 2017

Journal of Cell Science

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Akt is a key node in a range of signal transduction cascades and play a critical role in diseases such as cancer and diabetes. Fluorescentlytagged Akt reporters have been used to discern Akt localisation, yet it has not been clear how well these tools recapitulate the behaviour of endogenous Akt proteins. Here, we observed that fusion of eGFP to Akt2 impaired both its insulin-stimulated plasma membrane recruitment and its phosphorylation. Endogenous-like responses were restored by replacing eGFP with TagRFP-T. The improved response magnitude and sensitivity afforded by TagRFP-T-Akt2 over eGFP-Akt2 enabled monitoring of signalling outcomes in single cells at physiological doses of insulin with subcellular resolution and revealed two previously unreported features of Akt biology. In 3T3-L1 adipocytes, stimulation with insulin resulted in recruitment of Akt2 to the plasma membrane in a polarised fashion. Additionally, we observed oscillations in plasma membrane localised Akt2 in the presence of insulin with a consistent periodicity of 2 min. Our studies highlight the importance of fluorophore choice when generating reporter constructs and shed light on new Akt signalling responses that may encode complex signalling information.This article has an associated First Person interview with the first author of the paper.

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Section: Introductionmentioning

confidence: 99%

An improved Akt reporter reveals intra- and inter-cellular heterogeneity and oscillations in signal transduction

Norris

Yang

Krycer

et al. 2017

Journal of Cell Science

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“…At that time, as we used a conventional fluorescent microscope, it was impossible to reconstruct the images three-dimensionally, and the embryos were damaged by repeated fluorescent observations. In fact, it is widely believed that embryonic development is hampered by exposure to fluorescent or even room light wavelengths [16,17]. However, Squirrell et al [18] reported that they succeeded in obtaining pups after fluorescent imaging of embryos using two-photon laser scanning microscopy in the hamster.…”

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confidence: 99%

Long-Term, Six-Dimensional Live-Cell Imaging for the Mouse Preimplantation Embryo That Does Not Affect Full-Term Development

2009

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Abstract. Mammalian preimplantation embryonic development is achieved by tightly coordinated regulation of a great variety of temporal and spatial changes. Therefore, it would be valuable to analyze these events three-dimensionally and dynamically. We have previously developed a live-cell imaging method based on the expression of fluorescent proteins, using mRNA injection and time-lapse florescence microscopy. However, with conventional fluorescent microscopy, three-dimensional images could not be obtained due to the thickness of the embryos and the optical problem in which 'out-of focus blur' cannot be eliminated. Moreover, as the repeated exposure of intense excitation light to the cell yields phototoxicity, long-term observation was detrimental to embryonic development. Here, we improved our imaging system to enable six-dimensional live-cell imaging of mouse preimplantation embryos (x, y and z axes, time-lapse, multicolor and multisample). Importantly, by improving the imaging devices and optimizing the conditions for imaging, such as intensity of excitation and time intervals for image acquisition, the procedure itself was not detrimental to full-term development, although it is a prolonged imaging process. For example, live pups were obtained from embryos to which two different wavelengths of excitation (488 and 561 nm) were applied at 7.5-min intervals for about 70 h, and 51 images were acquired in the z axis at each time point; thus, a total of 56,814 fluorescent images were taken. All the pups were healthy, reproductively normal and not transgenic. Thus, this live-cell imaging technology is safe for full-term mouse development. This offers a novel approach for developmental and reproductive research in that it enables both retrospective and prospective analyses of development. It might also be applicable to assessment of embryo quality in fields such as human reproductive technology and production animal research. Key words: Embryo assessment, Full-term development, Live-cell imaging, Preimplantation embryo, Spinning-disk confocal microscopy (J. Reprod. Dev. 55: [343][344][345][346][347][348][349][350] 2009) ammalian preimplantation development is a process in which the union of two highly specialized gametes endows the resulting zygote with the potential to form undifferentiated cell types. This is followed by cell proliferation and the beginning of differentiation during the brief period leading up to implantation. In these processes, a number of cellular components and structures are regulated spatially and temporally, as seen in repeated cell division, cell cycle progression and genomic reprogramming via global epigenetic modifications [1]. Hence, many researchers have been attracted to these fundamental biological processes. However, the numbers of oocytes and embryos that can be collected are very limited, especially in mammals. Therefore, analysis of molecular mechanisms is hampered because of difficulties in conducting biochemical analyses on sufficient materials. Also, immunostaining methods ...

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“…The preferred method for labeling donor cells is the use of a reporter gene such as an enzyme or fluorescent protein. While these markers are not entirely without potential problems, including irregular expression (Swenson et al, 2007), possible cellular fusion (Terada et al, 2002), and physiological side effects (Devgan, et al, 2004;Guo et al, 2007;Huang et al, 2000;Liu et al, 1999), they allow detailed visualization of donor cell cytoarchitecture, and are remarkably stable and well-tolerated in many different cell types.…”

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confidence: 99%

Isolation of Progenitor Cells from GFP-Transgenic Pigs and Transplantation to the Retina of Allorecipients

Klassen

Warfvinge

Schwartz

et al. 2008

Cloning and Stem Cells

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Work in rodents has demonstrated that progenitor transplantation can achieve limited photoreceptor replacement in the mammalian retina; however, replication of these findings on a clinically relevant scale requires a large animal model. To evaluate the ability of porcine retinal progenitor cells to survival as allografts and integrate into the host retinal architecture, we isolated donor cells from fetal green fluorescent protein (GFP)-transgenic pigs. Cultures were propagated from the brain, retina, and corneo-scleral limbus. GFP expression rapidly increased with time in culture, although lower in conjunction with photoreceptor markers and glial fibrillary acid protein (GFAP), thus suggesting downregulation of GFP during differentiation. Following transplantation, GFP expression allowed histological visualization of integrated cells and extension of fine processes to adjacent plexiform layers. GFP expression in subretinal grafts was high in cells expressing vimentin and lower in cells expressing photoreceptor markers, again consistent with possible downregulation during differentiation. Cells survived transplantation to the injured retina of allorecipients at all time points examined (up to 10 weeks) in the absence of exogenous immune suppression without indications of rejection. These findings demonstrate the feasibility of allogeneic progenitor transplantation in a large mammal and the utility of the pig in ocular regeneration studies. 391

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Impact of embryonic expression of enhanced green fluorescent protein on early mouse development

Cited by 37 publications

References 18 publications

An improved Akt reporter reveals intra- and inter-cellular heterogeneity and oscillations in signal transduction

An improved Akt reporter reveals intra- and inter-cellular heterogeneity and oscillations in signal transduction

Long-Term, Six-Dimensional Live-Cell Imaging for the Mouse Preimplantation Embryo That Does Not Affect Full-Term Development

Isolation of Progenitor Cells from GFP-Transgenic Pigs and Transplantation to the Retina of Allorecipients

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