Work in the lab of E.S. is supported by the Swiss National Science Foundation. We would like to thank Dr. Beat Kunz for assistance with filming.

1. Preparation of RNAi Plasmid DNA for Cell Type-specific Gene Silencing
Plasmids (Figure 1) are synthesized using standard molecular cloning techniques, as previously described in detail3,4.
1.1 Cloning into the vectors: oligonucelotide design
<ol>
	<li>We use the same universal oligonucleotides and cloning protocols that are described in the product information provided with the pRFPRNAiC vector4 (ARK-Genomics).
	<ol class="la...

<ol>
	<li>Ch&#233;dotal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Further+tales+of+the+midline.">Further tales of the midline.</a> Current Opinion in Neurobiology. 21, 68-75 (2011).</li><li>Avraham, O., Hadas, Y., Vald, L., Zisman, S., Schejter, A., Visel, A., Klar, A. <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+axonal+guidance+and+sorting+in+dorsal+interneurons+by+the+Lim-HD+proteins+Lhx9+and+Lhx1.">Transcriptional control of axonal guidance and sorting in dorsal interneurons by the Lim-HD proteins Lhx9 and Lhx1.</a> Neural Development. 4, 21 (2009).</li><li>Wilson, N. H., Stoeckli, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+type+specific,+traceable+gene+silencing+for+functional+gene+analysis+during+vertebrate+neural+development.">Cell type specific, traceable gene silencing for functional gene analysis during vertebrate neural development.</a> Nucleic Acids Research. 39, e133 (2011).</li><li>Das, R. M., van Hateren, N. J., Howell, G. R., Farrell, E. R., Bangs, F. K., Porteous, V. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+robust+system+for+RNA+interference+in+the+chicken+using+a+modified+microRNA+operon.">A robust system for RNA interference in the chicken using a modified microRNA operon.</a> Developmental Biology. 294, 554-563 (2006).</li><li>Perrin, F. E., Stoeckli, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+lipophilic+dyes+in+studies+of+axonal+pathfinding+in+vivo.">Use of lipophilic dyes in studies of axonal pathfinding in vivo.</a> Microscopy Research and Technique. 48, 25-31 (2000).</li><li>Timmer, J., Johnson, J., Niswander, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+in+ovo+electroporation+for+the+rapid+analysis+of+neural-specific+murine+enhancers.">The use of in ovo electroporation for the rapid analysis of neural-specific murine enhancers.</a> Genesis. 29, 123-132 (2001).</li><li>Kieleczawa, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+modifications+of+the+standard+DNA+sequencing+protocol+allow+for+sequencing+through+siRNA+hairpins+and+other+repeats.">Simple modifications of the standard DNA sequencing protocol allow for sequencing through siRNA hairpins and other repeats.</a> Journal of Biomolecular Techniques. 16, 220-223 (2005).</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> Journal of Morphology. 88, 49-92 (1951).</li><li>Boulland, J., Halasi, G., Kasumacic, N., Glover, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenotransplantation+of+Human+Stem+Cells+into+the+Chicken+Embryo.">Xenotransplantation of Human Stem Cells into the Chicken Embryo.</a> J. Vis. Exp. (41), e2071 (2010).</li><li>Kim, B. G., Dai, H. -. N., McAtee, M., Vicini, S., Bregman, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Labeling+of+dendritic+spines+with+the+carbocyanine+dye+DiI+for+confocal+microscopic+imaging+in+lightly+fixed+cortical+slices.">Labeling of dendritic spines with the carbocyanine dye DiI for confocal microscopic imaging in lightly fixed cortical slices.</a> Journal of Neuroscience Methods. 162, 237-243 (2007).</li><li>Murphy, M. C., Fox, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anterograde+tracing+method+using+DiI+to+label+vagal+innervation+of+the+embryonic+and+early+postnatal+mouse+gastrointestinal+tract.">Anterograde tracing method using DiI to label vagal innervation of the embryonic and early postnatal mouse gastrointestinal tract.</a> Journal of Neuroscience Methods. 163, 213-225 (2007).</li><li>Helms, A. W., Abney, A. L., Ben-Arie, N., Zoghbi, H. Y., Johnson, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoregulation+and+multiple+enhancers+control+Math1+expression+in+the+developing+nervous+system.">Autoregulation and multiple enhancers control Math1 expression in the developing nervous system.</a> Development. 127, 1185-1196 (2000).</li><li>Li, X., Lufkin, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cre+recombinase+expression+in+the+floorplate,+notochord+and+gut+epithelium+in+transgenic+embryos+driven+by+the+Hoxa-1+enhancer+III.">Cre recombinase expression in the floorplate, notochord and gut epithelium in transgenic embryos driven by the Hoxa-1 enhancer III.</a> Genesis. 26, 121-122 (2000).</li><li>Mauti, O., Baeriswyl, T., Stoeckli, E. T. G. e. n. e. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silencing+by+Injection+and+Electroporation+of+dsRNA+in+Avian+Embryos.">Silencing by Injection and Electroporation of dsRNA in Avian Embryos.</a> Cold Spring Harbor Protocols. , (2008).</li><li>Krull, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+primer+on+using+in+ovo+electroporation+to+analyze+gene+function.">A primer on using in ovo electroporation to analyze gene function.</a> Developmental Dynamics. 229, 433-439 (2004).</li><li>Cullen, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancing+and+confirming+the+specificity+of+RNAi+experiments.">Enhancing and confirming the specificity of RNAi experiments.</a> Nature Methods. 3, 677-681 (2006).</li></ol>

No conflicts of interest declared.

This simple, vector-based artificial miRNA expression strategy can be used to knockdown endogenous gene expression in the chicken neural tube. These functional tools offer multiple gene silencing, temporal control and cell-type specificity, to facilitate the elucidation of complex developmental pathways. In particular, we have demonstrated the utility of these plasmids in commissural axon guidance, since the plasmids can be used to knockdown distinct genes in commissural neurons or in their intermediate target, th...

<table border="1">
 <tbody>
 <tr>
 <td>Name of reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>0.5 mm glass capillaries</td>
 <td>World Precision Instruments</td>
 <td>1B120F-4</td>
 </tr>
 <tr>
 <td>Glass needle puller</td>
 <td>Narishige</td>
 <td>PC-10</td>
 </tr>
 <tr>
 <td>Electroporator</td>
 <td>BTX</td>
 <td>ECM 830</td>
 </tr>
 <tr>
 <td>Sylgard silicone elastomer</td>
 <td>World Precision Instruments</td>
 <td>SYLG184</td>
 </tr>
 <tr>
 <td>Tungsten wire, 0.075 mm</td>
 <td>World Precision Instruments</td>
 <td>TGW0325</td>
 </tr>
 <tr>
 <td>Insect pins, 0.20 mm</td>
 <td>Fine Science Tools</td>
 <td>26002-20</td>
 </tr>
 <tr>
 <td>Insect pins, 0.10 mm</td>
 <td>Fine Science Tools</td>
 <td>26002-10</td>
 </tr>
 <tr>
 <td>Spring scissors</td>
 <td>Fine Science Tools</td>
 <td>15003-08</td>
 </tr>
 <tr>
 <td>Dumont #5 forceps</td>
 <td>Fine Science Tools</td>
 <td>11252-20</td>
 </tr>
 <tr>
 <td>Dumont #55 forceps</td>
 <td>Fine Science Tools</td>
 <td>11255-20</td>
 </tr>
 <tr>
 <td>Fast DiI</td>
 <td>Molecular Probes</td>
 <td>D-7756</td>
 </tr>
 <tr>
 <td>Fluorescent microscopes</td>
 <td>Olympus </td>
 <td>SZX12, BX51</td>
 </tr>
 </tbody>
</table>

in ovo electroporation of mirna-based plasmids in the developing neural tube and assessment of phenotypes by dii injection in open-book preparations

Commissural dI1 neurons have been extensively studied to elucidate the mechanisms underlying axon guidance during development1,2. These neurons are located in the dorsal spinal cord and send their axons along stereotyped trajectories. Commissural axons initially project ventrally towards and then across the floorplate. After crossing the midline, these axons make a sharp rostral turn and project longitudinally towards the brain. Each of these steps is regulated by the coordinated activities of attractive and repulsive guidance cues. The correct interpretation of these cues is crucial to the guidance of axons along their demarcated pathway. Thus, the physiological contribution of a particular molecule to commissural axon guidance is ideally investigated in the context of the living embryo. Accordingly, gene knockdown in vivo must be precisely controlled in order to carefully distinguish axon guidance activities of genes that may play multiple roles during development.
Here, we describe a method to knockdown gene expression in the chicken neural tube in a cell type-specific, traceable manner. We use novel plasmid vectors3 harboring cell type-specific promoters/enhancers that drive the expression of a fluorescent protein marker, followed directly by a miR30-RNAi transcript4 (located within the 3'-UTR of the cDNA encoding the fluorescent protein) (Figure 1). When electroporated into the developing neural tube, these vectors elicit efficient downregulation of gene expression and express bright fluorescent marker proteins to enable direct tracing of the cells experiencing knockdown3. Mixing different RNAi vectors prior to electroporation allows the simultaneous knockdown of two or more genes in independent regions of the spinal cord. This permits complex cellular and molecular interactions to be examined during development, in a manner that is fast, simple, precise and inexpensive. In combination with DiI tracing of commissural axon trajectories in open-book preparations5, this method is a useful tool for in vivo studies of the cellular and molecular mechanisms of commissural axon growth and guidance. In principle, any promoter/enhancer could be used, potentially making the technique more widely applicable for in vivo studies of gene function during development6.
This video first demonstrates how to handle and window eggs, the injection of DNA plasmids into the neural tube and the electroporation procedure. To investigate commissural axon guidance, the spinal cord is removed from the embryo as an open-book preparation, fixed, and injected with DiI to enable axon pathways to be traced. The spinal cord is mounted between coverslips and visualized using confocal microscopy.

Watch this Scientific Journal Video about In ovo Electroporation of miRNA-based Plasmids in the Developing Neural Tube and Assessment of Phenotypes by DiI Injection in Open-book Preparations at JoVE.c

In ovo Electroporation of miRNA-based Plasmids in the Developing Neural Tube and Assessment of Phenotypes by DiI Injection in Open-book Preparations

A method by which gene expression in the neural tube can be downregulated in a cell type-specific, traceable manner is described. We demonstrate how in ovo electroporation of microRNA-based plasmids that elicit spatiotemporally controlled RNA interference can be used to investigate commissural axon guidance in the developing neural tube.

In ovo Electroporation of miRNA-based Plasmids in the Developing Neural Tube and Assessment of Phenotypes by DiI Injection in Open-book Preparations

Commissural dI1 neurons have been extensively studied to elucidate the mechanisms underlying axon guidance during development1,2. These neurons are ...

in-ovo-electroporation-mirna-based-plasmids-developing-neural-tube

Research

JoVE Journal

Neuroscience

16.4K visualizações.  University of Zurich. Um método através do qual a expressão do gene no tubo neural pode ser downregulated em um tipo de célula específico forma rastreável é descrito. Demonstramos como In ovo Electroporação de microRNA baseados em plasmídeos que eliciam espaço-temporalmente controlada RNA de interferência pode ser utilizada para investigar a orientação do axónio comissural no tubo neural em desenvolvimento.

Vídeo: In ovo Electroporation of miRNA-based Plasmids in the Developing Neural Tube and Assessment of Phenotypes by DiI Injection in Open-book Preparations