Name: tracking cells in gfp-transgenic zebrafish using the photoconvertible psmorange system
Uploaded: 2016-02-05T15:00:00
Description: Watch this Scientific Journal Video about Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System at JoVE.com

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.

1. H2B-PSmOrange mRNA In Vitro Transcription and mRNA Purification
<ol>
	<li>Linearize the H2B-PSmOrange containing pCS2+ plasmid using the NotI restriction enzyme according to manufacturer&#39;s instructions. 
	Note: Perform the next steps using appropriate protections such as gloves and lab coat to prevent mRNA contamination and degradation.</li>
	<li>Purify the linearized DNA using a PCR Purification kit or phenol-chloroform based methods according to manufacturers&#39; protocols.</li>
	<li>Use 1 &#181...

<ol>
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Transgenic embryos carrying fluorescent reporters have helped fundamentally to understand embryonic development. However, there is still the essential need for promoters to facilitate the specific visualization of particular structures. In their absence, researchers rely on techniques such as the photoconversion of fluorescent proteins to find out about the origin and development of their structure of interest. This in turn is a crucial prerequisite to identify the molecular mechanisms involved in its development. Techni...

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.

<table border="0" cellpadding="0" cellspacing="0" width="821">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">PCR Purification Kit</td>
			<td style="width:233px;">Qiagen</td>
			<td align="right" style="width:119px;">28104</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mMESSAGE mMACHINE SP6 Transcription kit</td>
			<td>Ambion</td>
			<td>AM1340</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNeasy MiniElute Cleanup kit</td>
			<td>Qiagen</td>
			<td align="right">74204</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plastic Pasteur</td>
			<td>alpha laboratories</td>
			<td>LW4000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Original H2B-PSmOrange Plasmid</td>
			<td>Addgene</td>
			<td>31920</td>
			<td>The plasmid described in the paper is available in the Carl lab</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FemtoJet Microinjector</td>
			<td>Eppendorf</td>
			<td>5247 000.013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forceps (5 Inox)</td>
			<td>NeoLab</td>
			<td>2-1633</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lab-Tek II Chambered #1.5 German Coverglass System&#160;</td>
			<td>Nunc</td>
			<td align="right">155382</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nikon A1R+</td>
			<td>Nikon GmbH Germany</td>
			<td>No Number</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nikon PLAN Apo &#955; 20x air objective&#160;</td>
			<td>Nikon GmbH Germany</td>
			<td>No Number</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NIS Elements AR Software (v. 4.30.02)</td>
			<td>Nikon GmbH Germany/Laboratory Imaging</td>
			<td>No Number</td>
			<td></td>
		</tr>
	</tbody>
</table>

tracking cells in gfp-transgenic zebrafish using the photoconvertible psmorange system

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.

Figure 1 illustrates an example of the PSmOrange photoconversion system. The pineal complex is a conserved structure in the vertebrate dorsal diencephalon. Like in many other vertebrates, this complex consists of the pineal organ in the center of the diencephalon and the left-sided parapineal cells. Elegant but time-consuming uncaging experiments showed that parapineal cells originate in the anterior part of the pineal organ13. In tg(foxD3:GFP); tg(flh:GFP) tra...

Watch this Scientific Journal Video about Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System at JoVE.com

Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System

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.

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing ...

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