Thanks to the members of the Moens lab for helpful input during the writing of this article. H.K. is post-doctoral fellow supported by NIH/NICHD grant #5R01HD037909-08. C.B.M. is an investigator with the Howard Hughes Medical Institute.

A step-by-step guide to generating targeted chimeric zebrafish embryos by transplantation at the blastula or gastrula stage.
One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an organism that contains cells from more than one animal; mosaics are one type of chimera in which cells from more than one genotype are mixed, usually wild-type and mutant. In the zebrafish, chimeras can be readily made ...

<ol>
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The ease with which transplantation can be used to produce targeted chimeras is one of the great powers of the zebrafish as a vertebrate model. A modification of this protocol not described above allows analysis of maternal gene function for genes with essential roles in zygotic development. Since these mutant fish cannot survive until adulthood, it is necessary to transfer the mutant germline into an otherwise wild-type host embryo, creating &#x201C;germline clones&#x201D; of mutant cells. Generating germline mosaics in...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Injection rig</td>
<td>&nbsp;</td>
<td>Applied Scientific Instrumentation</td>
<td>MPPI-3</td>
<td>Foot pedal can be ordered separately</td>
</tr>
<tr>
<td>Pronase</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P5147</td>
<td>&#x201C;protease type XIV&#x201D;</td>
</tr>
<tr>
<td>Pen-Strep</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P4458</td>
<td>100x stock</td>
</tr>
<tr>
<td>Methyl cellulose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M-0387</td>
<td>&nbsp;</td>
<tr>
<td>Fluorescent dextrans</td>
<td>&nbsp;</td>
<td>Molecular Probes/ Invitrogen</td>
<td>various</td>
<td>Eg. lysine-fixable rhodamine dextran (D7162)</td>
</tr>
<tr>
<td>Injection pipettes (1.2mmx0.94mmx10cm)</td>
<td>&nbsp;</td>
<td>Sutter</td>
<td>BF120-94-10</td>
<td>&nbsp;</td>
<tr>
<td>Transplant pipette option 1</td>
<td>&nbsp;</td>
<td>World Precision Instruments</td>
<td>TW100-4</td>
<td>&nbsp;</td>
<tr>
<td>Transplant pipette option 2</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>#53508-400</td>
<td>&nbsp;</td>
<tr>
<td>micromanipulator</td>
<td>&nbsp;</td>
<td>Narishige</td>
<td>UMJ-3FC (?)</td>
<td>&nbsp;</td>
<tr>
<td>Micromanipulator magnetic stand</td>
<td>&nbsp;</td>
<td>Narishige</td>
<td>GJ-1</td>
<td>&nbsp;</td>
<tr>
<td>Transplant apparatus</td>
<td>&nbsp;</td>
<td>Sutter Instrument Co. </td>
<td>MI-10010</td>
<td>&nbsp;</td>
<tr>
<td>Fine micromanipulator</td>
<td>&nbsp;</td>
<td>Narishige</td>
<td>MMO-203</td>
<td>&nbsp;</td>
<tr>
<td>Depression slides</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>12560A</td>
<td>&nbsp;</td>
<tr>
<td>Agar molds for injection and transplants</td>
<td>&nbsp;</td>
<td>AL-AN Mfg, Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>
			<div>
			Reagents: Recipes for many of the reagents used for these procedures are found in The Zebrafish Book, which is available in full online at <a href="http://zfin.org/zf_info/zfbook/zfbk.html">http://zfin.org/zf_info/zfbook/zfbk.html</a>. Recipes are provided there for preparation of Fish Water, Embryo Medium with and without antibiotics, pronase for dechorionating, fluorescent dextrans, and methyl cellulose.		</div>

generating chimeric zebrafish embryos by transplantation

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an organism that contains cells from more than one animal; mosaics are one type of chimera in which cells from more than one genotype are mixed, usually wild-type and mutant. In the zebrafish, chimeras can be readily made by transplantation of cells from a donor embryo into a host embryo at the appropriate embryonic stage. Labeled donor cells are generated by injection of a lineage marker, such as a fluorescent dye, into the one-cell stage embryo. Labeled donor cells are removed from donor embryos and introduced into unlabeled host embryos using an oil-controlled glass pipette mounted on either a compound or dissecting microscope. Donor cells can in some cases be targeted to a specific region or tissue of the developing blastula or gastrula stage host embryo by choosing a transplantation site in the host embryo based on well-established fate maps.

Watch this Scientific Journal Video about Generating Chimeric Zebrafish Embryos by Transplantation at JoVE.com

Generating Chimeric Zebrafish Embryos by Transplantation

A step-by-step guide to generating targeted chimeric zebrafish embryos by transplantation at the blastula or gastrula stage.

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an ...

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21.0K visualizações.  Fred Hutchinson Cancer Research Center - FHCRC. Um guia passo-a-passo para a geração de embriões alvo zebrafish quimérico de transplante em fase de blástula ou gástrula.