Summary

在GFP-转基因斑马鱼使用Photoconvertible PSmOrange系统追踪单元

Published: February 05, 2016
doi:

Summary

We established the photoconvertible PSmOrange system as a powerful, straight-forward and cost inexpensive tool for in vivo cell tracking in GFP transgenic backgrounds. This protocol describes its application in the zebrafish model system.

Abstract

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing developmental processes as they happen in the living animal. Cells of interest can be labeled by using a tissue specific promoter to drive the expression of a fluorescent protein (FP) for the generation of transgenic lines. Using fluorescent photoconvertible proteins for this purpose additionally allows to precisely follow defined structures within the expression domain. Illuminating the protein in the region of interest, changes its emission spectrum and highlights a particular cell or cell cluster leaving other transgenic cells in their original color. A major limitation is the lack of known promoters for a large number of tissues in the zebrafish. Conversely, gene- and enhancer trap screens have generated enormous transgenic resources discretely labeling literally all embryonic structures mostly with GFP or to a lesser extend red or yellow FPs. An approach to follow defined structures in such transgenic backgrounds would be to additionally introduce a ubiquitous photoconvertible protein, which could be converted in the cell(s) of interest. However, the photoconvertible proteins available involve a green and/or less frequently a red emission state1 and can therefore often not be used to track cells in the FP-background of existing transgenic lines. To circumvent this problem, we have established the PSmOrange system for the zebrafish2,3. Simple microinjection of synthetic mRNA encoding a nuclear form of this protein labels all cell nuclei with orange/red fluorescence. Upon targeted photoconversion of the protein, it switches its emission spectrum to far red. The quantum efficiency and stability of the protein makes PSmOrange a superb cell-tracking tool for zebrafish and possibly other teleost species.

Introduction

Exponentially improving imaging techniques allow following developmental processes over time periods of up to about four consecutive days3. In zebrafish and many other animal model systems, specific cells, tissues, axonal or vascular structures are marked by transgenic green or sometimes red or yellow fluorescent proteins to facilitate visualization. However, in most transgenic lines the transgene is not specifically expressed in the cells of interest but also additional structures, which hinders the precise tracking of for instance single cells or groups of cells.

Fluorescent photoconvertible proteins are well suited for cell tracking during embryonic development. The prerequisites for the application of such proteins are a long-lived nature, a well-separated emission range upon conversion and bright fluorescence. Available photoconvertible proteins comprise those that change their emission range upon conversion such as Kaede4, KiGR5, mEos26, PS-CFP2 or Dendra27 and others which are only fluorescent when photoactivated (PAmCherry8, PAGFP9 or PATagRFP10). Their applications to track cells in existing FP-transgenic animals are however limited as they often involve a green fluorescent state or do not fulfill all of the above criteria. Only recently, Subach and colleagues reported the PSmOrange protein, which changes its emission from orange/red to far red upon photoconversion and was successfully applied in cells in culture and cultured cells injected into mice2.

To investigate the protein’s suitability for cell tracking in a living embryo, we generated an expression construct for the microinjection of nuclear-tagged H2B-PSmOrange into zebrafish embryos. We find that the protein fulfills all prerequisites for successful cell tracking in GFP transgenic backgrounds during the first 4 (and possibly more) days of zebrafish embryonic development. During this time, most of the cell migratory events are completed in fish making the PSmOrange system an excellent addition to the zebrafish toolkit.

Protocol

1. H2B-PSmOrange基因体外转录 mRNA纯化线性化使用根据制造商的说明的NotI限制性内切酶含有PCS2 +质粒H2B-PSmOrange。 注意:请使用适当的保护措施,如手套和白大褂,以防止污染的mRNA降解,并在接下来的步骤。 纯化使用根据制造商的协议的PCR纯化试剂盒或苯酚 – 氯仿基础的方法的线性化的DNA。 根据制造商的说明使用线性模板DNA 1微克了SP6-mRNA的转录。 通过在37℃?…

Representative Results

图1示出了PSmOrange光转换系统的一个例子。松果体复杂的是在脊椎动物背间脑的保守结构。像在许多其他脊椎动物中,此复合体由在间脑和左双面parapineal细胞的中心松果体器官。优雅但费时解笼锁的实验表明,parapineal细胞松果体器官13的前部产生。在TG(FOXD3:GFP); TG(FLH:GFP)转基因胚胎,无论松果体和parapineal细胞发育过程中的标签。为了评估PSmOra…

Discussion

携带荧光记者转基因胚胎有助于从根本上了解胚胎发育。然而,仍然有启动子的基本需要,以促进特定结构的特定的可视化。在他们的缺席,研究人员依靠技术,如荧光蛋白的光转化,了解他们的利益结构的起源和发展。这又是鉴定参与在其发展的分子机制的关键先决条件。在斑马鱼领域的技术进步,现在允许更换FP在方法14,15使用CRISPR-Cas9介导的敲除转基因系与,例如,photoconvertible蛋白?…

Declarações

The authors have nothing to disclose.

Acknowledgements

We thank O. Subach for providing the original H2B-PSmOrange plasmid and our fish facility team for fish care. We are grateful to the Nikon Imaging Center at the University of Heidelberg for access to microscopy equipment and analysis software. We acknowledge the support of the Core Facility Live Cell Imaging Mannheim at the CBTM (DFG INST 91027/10-1 FUGG). This work was supported by the Excellenzcluster CellNetworks, EcTop Spatio-temporal coordination of signaling processes (EcTop 2), University of Heidelberg to C.A.B. and the Medical Faculty Mannheim of the University Heidelberg and the DFG (FOR 1036/2, 298/3-1 and 298/6-1) to M.C.

Materials

PCR Purification Kit Qiagen 28104
mMESSAGE mMACHINE SP6 Transcription kit Ambion AM1340
RNeasy MiniElute Cleanup kit Qiagen 74204
Plastic Pasteur alpha laboratories LW4000
Original H2B-PSmOrange Plasmid Addgene 31920 The plasmid described in the paper is available in the Carl lab
FemtoJet Microinjector Eppendorf 5247 000.013
Forceps (5 Inox) NeoLab 2-1633
Lab-Tek II Chambered #1.5 German Coverglass System  Nunc 155382
Nikon A1R+ Nikon GmbH Germany No Number
Nikon PLAN Apo λ 20x air objective  Nikon GmbH Germany No Number
NIS Elements AR Software (v. 4.30.02) Nikon GmbH Germany/Laboratory Imaging No Number

Referências

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Beretta, C. A., Dross, N., Engel, U., Carl, M. Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System. J. Vis. Exp. (108), e53604, doi:10.3791/53604 (2016).

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