Labelling cell structures and tracking cell lineage in zebrafish using SNAP-tag

Campos, Cláudia; Kamiya, Mako; Banala, Sambashiva; Johnsson, Kai; González‐Gaitán, Marcos

doi:10.1002/dvdy.22574

Cited by 31 publications

(24 citation statements)

References 82 publications

(73 reference statements)

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“…However, this strategy is easily adaptable to other proteins (e.g. Figure 3 D) as well, and similar strategies have been used by us and other investigators, in a range of organisms and for different applications (Jansen et al, 2007;Erhardt et al, 2008;McMurray and Thorner, 2008;Maduzia et al, 2010;Bojkowska et al, 2011;Campos et al, 2011;Dunleavy et al, 2011;Silva et al, in press; also reviewed in O' Hare et al, 2007). We will describe two typical types of SNAP-labeling strategies: Pulse-Chase (Basic Protocol 1) and Quench-Chase-Pulse (Basic Protocol 2), which allow for the analysis of old and new protein pools, respectively.…”

Section: Introductionmentioning

confidence: 82%

Analysis of Protein Turnover by Quantitative SNAP‐Based Pulse‐Chase Imaging

et al. 2012

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Assessment of protein dynamics in living cells is crucial for understanding their biological properties and functions. The SNAP‐tag, a self labeling suicide enzyme, presents a tool with unique features that can be adopted for determining protein dynamics in living cells. Here we present detailed protocols for the use of SNAP in fluorescent pulse‐chase and quench‐chase‐pulse experiments. These time‐slicing methods provide powerful tools to assay and quantify the fate and turnover rate of proteins of different ages. We cover advantages and pitfalls of SNAP‐tagging in fixed‐ and live‐cell studies and evaluate the recently developed fast‐acting SNAPf variant. In addition, to facilitate the analysis of protein turnover datasets, we present an automated algorithm for spot recognition and quantification. Curr. Protoc. Cell Biol. 55:8.8.1‐8.8.34. © 2012 by John Wiley & Sons, Inc.

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Section: Introductionmentioning

confidence: 82%

Analysis of Protein Turnover by Quantitative SNAP‐Based Pulse‐Chase Imaging

et al. 2012

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“…It is derived from the human DNA repair protein O 6 -alkylguanine-DNA alkyltransferase (hAGT) and can covalently react with O 6 -benzylguanine (BG) derivatives bearing a chemical probe in living cells (22). This method was used to label several subcellular compartments of zebrafish embryos including the nucleus in conjugation with a variety of fluorophore dyes (26). Furthermore, the same technique was also used for live-cell direct stochastic optical reconstruction microscopy (dSTORM) imaging of histone H2B protein fused with SNAP tag (27).…”

Section: Figure 1 (A) Schematic Representation Of Talen-mediated Genmentioning

confidence: 99%

Single-Molecule Fluorescence Microscopy Methods in Chromatin Biology

Tatavosian

Zhen

Ren

2015

ACS Symposium Series

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Classical biochemical and molecular genetic techniques have provided fundamental insights into genomic functions and chromatin organization. Recent advances in the development of fluorescence microscopy imaging methods as well as new labeling techniques enable visualizing chromatin structure and tracking dynamics of transcription factors within mammalian cells at a single-molecule level. Single particle tracking of transcription factors within cells provides insight into how transcription factors explore the complex nuclear environment and assemble on their target sites. Superresolution microscopy based on single-molecule centroid determination has been applied to map chromatin structure at a nucleosomal resolution. This chapter outlines recent advances in the application of single molecule-based fluorescence microscopy techniques in chromatin biology within mammalian cells.

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“…In addition to autofluorescent protein genes, research in zebrafish also makes use of small molecular fluorescent probes. For instance, common use is made of Alexa dyes (Life Technologies) for whole mount in situ fluorescence hybridization (Clay and Ramakrishnan, 2005;Welten et al, 2006;Brend and Holley, 2009) or immunohistochemistry (Campos et al, 2011).…”

Section: Fluorescent Readout Technologiesmentioning

confidence: 99%

Zebrafish embryos and larvae: A new generation of disease models and drug screens

Ali

Champagne

Spaink

et al. 2011

Birth Defects Research Part C: Embryo Today: Reviews

215

135

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Technological innovation has helped the zebrafish embryo gain ground as a disease model and an assay system for drug screening. Here, we review the use of zebrafish embryos and early larvae in applied biomedical research, using selected cases. We look at the use of zebrafish embryos as disease models, taking fetal alcohol syndrome and tuberculosis as examples. We discuss advances in imaging, in culture techniques (including microfluidics), and in drug delivery (including new techniques for the robotic injection of compounds into the egg). The use of zebrafish embryos in early stages of drug safety-screening is discussed. So too are the new behavioral assays that are being adapted from rodent research for use in zebrafish embryos, and which may become relevant in validating the effects of neuroactive compounds such as anxiolytics and antidepressants. Readouts, such as morphological screening and cardiac function, are examined. There are several drawbacks in the zebrafish model. One is its very rapid development, which means that screening with zebrafish is analogous to "screening on a run-away train." Therefore, we argue that zebrafish embryos need to be precisely staged when used in acute assays, so as to ensure a consistent window of developmental exposure. We believe that zebrafish embryo screens can be used in the pre-regulatory phases of drug development, although more validation studies are needed to overcome industry scepticism. Finally, the zebrafish poses no challenge to the position of rodent models: it is complementary to them, especially in early stages of drug research.

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Labelling cell structures and tracking cell lineage in zebrafish using SNAP-tag

Cited by 31 publications

References 82 publications

Analysis of Protein Turnover by Quantitative SNAP‐Based Pulse‐Chase Imaging

Analysis of Protein Turnover by Quantitative SNAP‐Based Pulse‐Chase Imaging

Single-Molecule Fluorescence Microscopy Methods in Chromatin Biology

Zebrafish embryos and larvae: A new generation of disease models and drug screens

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