Name: in ovo electroporation in embryonic chick retina
Uploaded: 2012-02-05T13:00:00
Description: Watch this Scientific Journal Video about In Ovo Electroporation in Embryonic Chick Retina at JoVE.com

We would like to thank Mr. Maaz Enver for the use of his HD camera (Cannon VIXIA HFS100). This work was supported in part by grants from NIH (EY018738), New Jersey Commission on Spinal Cord Research (08-3074-SCR-E-0 and 10-3091-SCR-E-0), and Busch Biomedical Research Award.

Since some parts of the protocol are performed as previously described in other JoVE videos2 and papers3 with minor modification we will not discuss those in detail here.
1. Egg Handling and Needle Preparation
<ol>
 <li>Eggs can be stored in a wine cooler at about 13&deg;C for up to 1 week. If the temperature is too high, embryos will start to develop abnormally, while lower temperature causes high mortality. Once you are ready to incubate take out the eggs from the wi...

<ol>
	<li>Doh, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+retinal+cell+development+in+chick+embryo+by+immunohistochemistry+and+in+ovo+electroporation+techniques.">Analysis of retinal cell development in chick embryo by immunohistochemistry and in ovo electroporation techniques.</a> BMC. Dev. Biol. 10, (2010).</li><li>Blank, M. C., Chizhikov, V., Millen, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Ovo+Electroporations+of+HH+Stage+10+Chicken+Embryos.">In Ovo Electroporations of HH Stage 10 Chicken Embryos.</a> J. Vis. Exp. (9), e408-e408 (2007).</li><li>Nakamura, H., Funahashi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+of+DNA+into+chick+embryos+by+in+ovo+electroporation.">Introduction of DNA into chick embryos by in ovo electroporation.</a> Methods. 24, 43-48 (2001).</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.+1951.">A series of normal stages in the development of the chick embryo. 1951.</a> Dev. Dyn.. 195, 231-272 (1992).</li><li>Hamburger, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+stage+series+of+the+chick+embryo.">The stage series of the chick embryo.</a> Dev. Dyn. 195, 273-275 (1992).</li></ol>

Targeted retinal injection and in ovo electroporation in embryonic retina at E4 stage can specifically target retinal progenitor cells resulting in the ability to visualize all six major retina cell types at the single cell level. In ovo electroporation at HH10 (~E1.5) targeting the optic vesicle is able to transfect cells that develop to form the eye. However, these cells have a very high turnover at this time and this method is not specific for retinal cells. It may be that the high cell turnover rate...

<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 pathogen-free (SPF) white leghorn 			chicken eggs</td>
 <td>Sunrise 			Farms (Catskill, NY)</td>
 <td>&nbsp;
 </td>
 <td>&nbsp;
 </td>
 </tr>
 <tr>
 <td>Chicken egg incubator</td>
 <td>GQF 			manufacturing, Savannah, GA</td>
 <td>Model-1550
 </td>
 <td>Set to 60% humidity and 37.5&deg;C.</td>
 </tr>
 <tr>
 <td>Glass capillary tubes</td>
 <td>World 			Precision Instrument Inc.</td>
 <td>TW150F-4</td>
 <td>&nbsp;
 </td>
 </tr>
 <tr>
 <td>0.1 ml syringe</td>
 <td>Hamilton 			Co. Reno, Nevada</td>
 <td>Gastight 			1710</td>
 <td>Alternatively 1ml syringe can be used as well</td>
 </tr>
 <tr>
 <td>Masterflex Silicone (peroxide) Tubing</td>
 <td>Cole-Parmer</td>
 <td>HV-96400-13</td>
 <td>Cut a small piece (1 cm) for attaching the glass 			needle to the syringe</td>
 </tr>
 <tr>
 <td>Tweezers</td>
 <td>Dumont</td>
 <td>AA</td>
 <td>&nbsp;
 </td>
 </tr>
 <tr>
 <td>Micromanipulator</td>
 <td>World 			Precision Instrument Inc.</td>
 <td>M3301-M3</td>
 <td>&nbsp;
 </td>
 </tr>
 <tr>
 <td>Plasmid DNA</td>
 <td>&nbsp;
 </td>
 <td>&nbsp;
 </td>
 <td>pCAG-GFP was borrowed from Dr. Connie Cepko</td>
 </tr>
 <tr>
 <td>Fast Green FCF</td>
 <td>Sigma-Aldrich</td>
 <td>F7252</td>
 <td>Dilute it to 0.025 % with PBS</td>
 </tr>
 <tr>
 <td>Pulse generator</td>
 <td>Harvard 			Apparatus, MA</td>
 <td>BTX 			ECM 830</td>
 <td>Square wave generator</td>
 </tr>
 <tr>
 <td>Electrodes</td>
 <td>Harvard Apparatus, MA</td>
 <td>BTX 			model 514</td>
 <td>Our electrodes were spaced 3-5 mm apart</td>
 </tr>
 <tr>
 <td>Monochrome Digital Camera</td>
 <td>Zeiss, Germany</td>
 <td>Axiocam 			MRM</td>
 <td>&nbsp;
 </td>
 </tr>
 <tr>
 <td>Fluorescent Dissection Microscope</td>
 <td>Leica Microsystems, Germany</td>
 <td>Leica 			MZ16FA</td>
 <td>&nbsp;
 </td>
 </tr>
 <tr>
 <td>Upright Fluorescence
 Microscope</td>
 <td>Zeiss, Germany</td>
 <td>Zeiss 			Axio Imager A1</td>
 <td>&nbsp;
 </td>
 </tr>
 </tbody>
</table>

in ovo electroporation in embryonic chick retina

Chicken embryonic retina is an excellent tool to study retinal development in higher vertebrates. Because of large size and external development, it is comparatively very easy to manipulate the chick embryonic retina using recombinant DNA/RNA technology. Electroporation of DNA/RNA constructs into the embryonic retina have a great advantage to study gene regulation in retinal stem/progenitor cells during retinal development. Different type of assays such as reporter gene assay, gene over-expression, gene knock down (shRNA) etc. can be performed using the electroporation technique. This video demonstrates targeted retinal injection and in ovo electroporation into the embryonic chick retina at the Hamburger and Hamilton stage 22-23, which is about embryonic day 4 (E4). Here we show a rapid and convenient in ovo electroporation technique whereby a plasmid DNA that expresses green fluorescent protein (GFP) as a marker is directly delivered into the chick embryonic subretinal space and followed by electric pulses to facilitate DNA uptake by retinal stem/progenitor cells. The new method of retinal injection and electroporation at E4 allows the visualization of all retinal cell types, including the late-born neurons1, which has been difficult with the conventional method of injection and electroporation at E1.52.

Watch this Scientific Journal Video about In Ovo Electroporation in Embryonic Chick Retina at JoVE.com

In Ovo Electroporation in Embryonic Chick Retina

The overall goal of this video is to show how to perform targeted retinal injection and in ovo electroporation of DNA/RNA constructs into the chick embryonic retina at the Hamburger and Hamilton stage 22-23, which is about embryonic day 4 (E4). This technique is very useful to study gene expression, gene regulation, and morphological change in developing chick retina.

In Ovo Electroporation in Embryonic Chick Retina

Chicken embryonic retina is an excellent tool to study retinal development in higher vertebrates. Because of large size and external development, it is ...

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