We thank Dr. Takahiko Matsuda for providing the pCAG-mCherry construct. YCM is supported by a Career Development Award from the Schweppe Foundation and a grant from the Whitehall Foundation.

1. Supply Preparation (not in video)
<ol>
 <li>Prepare a fresh 1X dilution of Penicillin/Streptomycin (Pen/Strep) in 1X PBS. No more than 50 mL is necessary. Filter with a 0.22 &mu;m syringe filter.</li>
 <li>Prepare injection constructs by combining desired plasmid constructs. We put all constructs in the pCAG-Flag vector with appropriate stoichiometry to a final volume of ~10 &mu;L. Also, pCAG-GFP14 or pCAG-mCherry should be added for tracking electroporation efficiency and electroporated cells. We use pCA...

<ol>
	<li>Wightman, R. M., Robinson, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+changes+in+mesolimbic+dopamine+and+their+association+with+'reward'.">Transient changes in mesolimbic dopamine and their association with 'reward'.</a> J. Neurochem. 82, 721-735 (2002).</li><li>Kelley, A. E., Berridge, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neuroscience+of+natural+rewards:+relevance+to+addictive+drugs.">The neuroscience of natural rewards: relevance to addictive drugs.</a> J. Neurosci. 22, 3306-3311 (2002).</li><li>Moore, D. J., West, A. B., Dawson, V. L., Dawson, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+pathophysiology+of+Parkinson's+disease.">Molecular pathophysiology of Parkinson's disease.</a> Annu. Rev. Neurosci. 28, 57-87 (2005).</li><li>Dailly, E., Chenu, F., Renard, C. E., Bourin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine,+depression+and+antidepressants.">Dopamine, depression and antidepressants.</a> Fundam. Clin. Pharmacol. 18, 601-607 (2004).</li><li>Sesack, S. R., Carr, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+prefrontal+cortex+inputs+to+dopamine+cells:+implications+for+schizophrenia.">Selective prefrontal cortex inputs to dopamine cells: implications for schizophrenia.</a> Physiol. Behav. 77, 513-517 (2002).</li><li>Ang, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+control+of+midbrain+dopaminergic+neuron+development.">Transcriptional control of midbrain dopaminergic neuron development.</a> Development. 133, 3499-3506 (2006).</li><li>Andersson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+intrinsic+determinants+of+midbrain+dopamine+neurons.">Identification of intrinsic determinants of midbrain dopamine neurons.</a> Cell. 124, 393-405 (2006).</li><li>Hamburger, V., Hamilton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Series+of+Normal+Stages+in+the+Development+of+the+Chick+Embryo.">A Series of Normal Stages in the Development of the Chick Embryo.</a> J. Morphol. 88, 49 (1951).</li><li>Bellairs, R., Osmond, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The atlas of chick development. , (2005).</li><li>Itasaki, N., Bel-Vialar, S., Krumlauf, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term='Shocking'+developments+in+chick+embryology:+electroporation+and+in+ovo+gene+expression.">'Shocking' developments in chick embryology: electroporation and in ovo gene expression.</a> Nat. Cell Biol. 1, 203-207 (1999).</li><li>Momose, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+targeting+of+gene+expression+in+chick+embryos+by+microelectroporation.">Efficient targeting of gene expression in chick embryos by microelectroporation.</a> Dev. Growth Differ. 41, 335-344 (1999).</li><li>Muramatsu, T., Mizutani, Y., Ohmori, Y., Okumura, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+three+nonviral+transfection+methods+for+foreign+gene+expression+in+early+chicken+embryos+in+ovo.">Comparison of three nonviral transfection methods for foreign gene expression in early chicken embryos in ovo.</a> Biochem. Biophys. Res. Commun. 230, 376-380 (1997).</li><li>Nishi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-efficiency+in+vivo+gene+transfer+using+intraarterial+plasmid+DNA+injection+following+in+vivo+electroporation.">High-efficiency in vivo gene transfer using intraarterial plasmid DNA injection following in vivo electroporation.</a> Cancer Res. 56, 1050-1055 (1996).</li><li>Matsuda, T., Cepko, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+and+RNA+interference+in+the+rodent+retina+in+vivo+and+in+vitro.">Electroporation and RNA interference in the rodent retina in vivo and in vitro.</a> Proceedings of the National Academy of Sciences of the United States of America. 101, 16-22 (2004).</li><li>Blank, M. C., Chizhikov, V., Millen, K. 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The authors have no potential conflicting interests to disclose.

The in ovo electroporation of embryonic chick midbrain offers a low cost and rapid alternative to the generation of transgenic or knockout animals to perform in vivo study of gene functions in midbrain dopaminergic neuron development. Using short 2 mm long L-shaped platinum electrodes together with embryonic midbrain-specific DNA injection is the key for achieving efficient expression of the gene of interest in midbrain dopaminergic neuron progenitors. In addition, using correct HH stage 11 chick embryo...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Comments </td>
		</tr>
		<tr>
			<td>Fertilized chicken eggs</td>
			<td>Charles River Laboratories</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Egg incubator</td>
			<td>GQF Manufacturing</td>
			<td>1502 Sportsman</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>BTX Electroporator</td>
			<td>BTX Harvard Apparatus</td>
			<td>ECM830</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Electrodes</td>
			<td>BTX Harvard Apparatus</td>
			<td>45-0162 L-shaped genetrodes</td>
			<td>For use with ECM830 electroporator</td>
		</tr>
		<tr>
			<td>Platinum iridium wire</td>
			<td>Alfa Aesar</td>
			<td>#10056</td>
			<td>0.5 mm diameter 
			For making the 2 mm long electrodes</td>
		</tr>
		<tr>
			<td>Glass capillary tube</td>
			<td>World Precision Instrument</td>
			<td>TW100F-4</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microloader pipette tip</td>
			<td>Eppendorf</td>
			<td>5242 956.003</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>India ink</td>
			<td>Staples</td>
			<td>&nbsp;</td>
			<td>Filter 20% before use</td>
		</tr>
		<tr>
			<td>Microinjector</td>
			<td>Parker Automation, 
			Parker Hannifin Cooperation</td>
			<td>Picospritzer III</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fluorescent dissection microscope</td>
			<td>Leica</td>
			<td>MZ16F</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Sutter Instrument</td>
			<td>D-97</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

in ovo electroporation in chick midbrain for studying gene function in dopaminergic neuron development

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain dopaminergic neurons is implicated in Parkinson's disease, Schizophrenia, depression, and dementia1-5. Thus, studying the regulation of midbrain dopaminergic neuron differentiation could not only provide important insight into mechanisms regulating midbrain development and neural progenitor fate specification, but also help develop new therapeutic strategies for treating a variety of human neurological disorders.
Dopaminergic neurons differentiate from neural progenitors lining the ventricular zone of embryonic ventral midbrain. The development of neural progenitors is controlled by gene expression programs6,7. Here we report techniques utilizing electroporation to express genes specifically in the midbrain of Hamburger Hamilton (HH) stage 11 (thirteen somites, 42 hours) chick embryos8,9. The external development of chick embryos allows for convenient experimental manipulations at specific embryonic stages, with the effects determined at later developmental time points10-13. Chick embryonic neural tubes earlier than HH stage 13 (nineteen somites, 48 hours) consist of multipotent neural progenitors that are capable of differentiating into distinct cell types of the nervous system. The pCAG vector, which contains both a CMV promoter and a chick &beta;-actin enhancer, allows for robust expression of Flag or other epitope-tagged constructs in embryonic chick neural tubes14. In this report, we emphasize special measures to achieve regionally restricted gene expression in embryonic midbrain dopaminergic neuron progenitors, including how to inject DNA constructs specifically into the embryonic midbrain region and how to pinpoint electroporation with small custom-made electrodes. Analyzing chick midbrain at later stages provides an excellent in vivo system for plasmid vector-mediated gain-of-function and loss-of-function studies of midbrain development. Modification of the experimental system may extend the assay to other parts of the nervous system for performing fate mapping analysis and for investigating the regulation of gene expression.

Watch this Scientific Journal Video about In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development at JoVE.com

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

To assess the function and the regulation of genes during the development of midbrain dopaminergic neurons, we describe a method that involves in ovo electroporation of plasmid DNA constructs into embryonic chick ventral midbrain dopaminergic neuron progenitors. This technique can be used to achieve efficient expression of genes of interest to study different aspects of midbrain development and dopaminergic neuron differentiation.

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain ...

in-ovo-electroporation-chick-midbrain-for-studying-gene-function

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JoVE Journal

Neuroscience

11.3K vistas.  Children's Hospital of Chicago Research Center. Para evaluar la función y la regulación de los genes durante el desarrollo de las neuronas del cerebro medio dopaminérgico, se describe un método que consiste en In ovo Electroporación de construcciones de ADN plásmido en células progenitoras embrionarias de pollo ventral del cerebro medio de neuronas dopaminérgicas. Esta técnica puede utilizarse para lograr la expresión eficiente de genes de interés para estudiar diferentes aspectos del desarrol...