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>
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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 ビュー.  University of Zurich. 神経管における遺伝子発現は、細胞型特異的、トレーサブルな方法でダウンレギュレートすることができる方法が記載されている。我々はどのように実証するエレクトロポレーションは、途上神経管における交連軸索ガイダンスを調査するために使用することが?...

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