The work was supported by Barncancerfonden (PR2013-0104), Swedish Research Council (12187-18-3), &#246;gonfonden, Kronprinsessan Margaretas arbetsn&#228;mnd f&#246;r synskadade, Synfr&#228;mjandets forskningsfond and St Eriks &#246;gonsjukhus forskningsstipendier.

This protocol is performed in accordance with the recommendations in the &#8220;Guide for the Care and Use of Laboratory Animals of the Association for research in vision and ophthalmology&#8221;.
1. Egg Handling and Eye collection
<ol>
	<li>Store white Leghorn eggs at 12-14 &#176;C for no longer than 1 week. If available, use a refrigerated wine cooler to store the fertilized eggs. 
	Note: Higher store temperatures lead to abnormal development of embryos, while lower temperatures incre...

<ol>
	<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 Commu. 230, 376-380 (1997).</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> Method. 24, 43-48 (2001).</li><li>Kohl, A., Hadas, Y., Klar, A., Sela-Donenfeld, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+patterns+and+targets+of+dA1+interneurons+in+the+chick+hindbrain.">Axonal patterns and targets of dA1 interneurons in the chick hindbrain.</a> J Neurosc. 32, 5757-5771 (2012).</li><li>Islam, M. M., Doh, S. T., Cai, L. <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+electroporation+in+embryonic+chick+retina.">In ovo electroporation in embryonic chick retina.</a> J Vis Exp. , (2012).</li><li>Sparrow, J. R., Hicks, D., Barnstable, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+commitment+and+differentiation+in+explants+of+embryonic+rat+neural+retina.+Comparison+with+the+developmental+potential+of+dissociated+retina.">Cell commitment and differentiation in explants of embryonic rat neural retina. Comparison with the developmental potential of dissociated retina.</a> Brain res Dev brain re. 51, 69-84 (1990).</li><li>Tomita, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+hairy+and+Enhancer+of+split+homolog+1+regulates+differentiation+of+retinal+neurons+and+is+essential+for+eye+morphogenesis.">Mammalian hairy and Enhancer of split homolog 1 regulates differentiation of retinal neurons and is essential for eye morphogenesis.</a> Neuro. 16, 723-734 (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> Proc Nat Acad Sci US. 101, 16-22 (2004).</li><li>Donovan, S. L., Dyer, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+square+wave+electroporation+of+retinal+explant+cultures.">Preparation and square wave electroporation of retinal explant cultures.</a> Nature protocol. 1, 2710-2718 (2006).</li><li>Thangaraj, G., Greif, A., Layer, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+explant+culture+of+the+embryonic+chicken+retina+with+long-term+preservation+of+photoreceptors.">Simple explant culture of the embryonic chicken retina with long-term preservation of photoreceptors.</a> Exp eye re. 93, 556-564 (2011).</li><li>Shirazi Fard, S., All-Ericsson, C., Hallbook, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+heterogenic+final+cell+cycle+of+chicken+retinal+Lim1+horizontal+cells+is+not+regulated+by+the+DNA+damage+response+pathway.">The heterogenic final cell cycle of chicken retinal Lim1 horizontal cells is not regulated by the DNA damage response pathway.</a> Cell Cycl. 13, 408-417 (2014).</li><li>Shirazi Fard, S., Thyselius, M., All-Ericsson, C., Hallbook, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+terminal+basal+mitosis+of+chicken+retinal+Lim1+horizontal+cells+is+not+sensitive+to+cisplatin-induced+cell+cycle+arrest.">The terminal basal mitosis of chicken retinal Lim1 horizontal cells is not sensitive to cisplatin-induced cell cycle arrest.</a> Cell Cycl. 13, 3698-3706 (2014).</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, 49-92 (1951).</li><li>Gavet, O., Pines, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+cyclin+B1-Cdk1+synchronizes+events+in+the+nucleus+and+the+cytoplasm+at+mitosis.">Activation of cyclin B1-Cdk1 synchronizes events in the nucleus and the cytoplasm at mitosis.</a> J Cell Bio. 189, 247-259 (2010).</li><li>Sauer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitosis+in+the+neural+tube.">Mitosis in the neural tube.</a> J Comp Neurol. 62, 377-405 (1935).</li><li>Baye, L. M., Link, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interkinetic+nuclear+migration+and+the+selection+of+neurogenic+cell+divisions+during+vertebrate+retinogenesis.">Interkinetic nuclear migration and the selection of neurogenic cell divisions during vertebrate retinogenesis.</a> J Neurosc. 27, 10143-10152 (2007).</li></ol>

In this work a detailed protocol for dissection, electroporation or chemical treatment, and culturing of whole retinal explants from chicken embryos is presented. This protocol is easy, quick and allows for both a high success rate and reproducible results.
Electroporation of whole retinal explants produces large areas of cells that express the gene construct of interest. It is easy to correctly position the electrodes and to expose a specific portion of the retina to a define...

The retina is part of the central nervous system and it is, with its relative simplicity and well-characterized cellular architecture, a popular model for studying central nervous system development. The eye of the chicken embryo is relatively large in comparison to the rest of the embryo. It is therefore easily accessible in ovo for experimental manipulations, such as injections or electroporation, and serves as an excellent tool to gain knowledge about retinal cell and developmental biology in vivo. Despite these major advantages, survival of the embryos can be low when experiments are invasive such as with electroporations, repeated injections, or combined experimental manipulations.
Electroporation of DNA plasmids into the chicken embryo in ovo is an important and well-established technique1. It allows for labeling of neurons, tracing of cell fate as well as neuronal tracts in the central nervous system and it allows for ectopic gene expression to analyze protein function in vivo. The technique has been used for studies of neural tube2, hindbrain3, and retina4. Electroporation of embryonic retina in ovo has some experimental difficulties that are related to the in vivo situation. The position of the eye, due to the cranial folding of the embryo, is relatively close to the heart. This proximity increases the risk of cardiac arrest following electroporation, and the risk increases with the age of the embryo. Moreover, to access the eye, it is necessary to open the embryonic membranes, thereby increasing the risk for bleeding, malformations and subsequent reduced viability. When testing and optimizing a new DNA plasmid often without a known phenotypic outcome, these limitations may decrease the efficacy and power of the method even for an experienced experimentalist. As presented in this protocol, the culture of the whole retinal explant, defined as the whole neural retina with the pigment epithelium removed, is an efficient method that complements the in ovo approach.
Intraocular injections of chemical reagents are relatively easy to perform in ovo. However, the effective and exact concentration of injected reagents within the neural retina may be difficult to fully control. The injected volume may vary due to leakage and the exact site of injection may affect both the distribution of the reagent within the eye and the diffusion through the vitreous body. The variability will have major implications for the interpretation of the results when i.e., a dose response curve for an enzyme inhibitor is determined; particularly if the effect is small and the temporal window of the effect is narrow. Moreover, only a single eye can be used from each embryo when performing in ovo experiments due to potential systemic effects via the blood stream onto the contralateral eye. Age matching is important when studying development and the individual variability between treated and control embryos may lead to additional experimental variability.
For these reasons, an ex ovo method based on retinal explants from chicken embryos was developed, in which the neural retina can be exposed to a uniform and controlled experimental condition in vitro. The present protocol was developed based on previous protocols5-9. Retinal explants from stage (st) 20 (embryonic days [E] 3) to st31 (E7) chicken embryos were dissected, cultured and electroporated with a defined DNA plasmid concentration or exposed to a medium containing a defined concentration of a chemical reagent. The protocol presented here has been successfully implemented in recent publications, using several different chemical reagents, including regulators of the DNA damage pathway, such as KU55933, SB 218078, and NSC 109555 ditosylate, and the cell cycle, such as Cdk1/2 inhibitor III10,11.

<table border="0" cellpadding="0" cellspacing="0" width="859">
	<tbody>
		<tr>
			<td>1xPBS (tablet)</td>
			<td>Life technologies</td>
			<td>18912-014</td>
			<td></td>
		</tr>
		<tr>
			<td>10xDPBS</td>
			<td>Life technologies</td>
			<td>14080-048</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm Petri dish</td>
			<td>VWR</td>
			<td>734-0006</td>
			<td></td>
		</tr>
		<tr>
			<td>100 &#956;L pipette tips</td>
			<td>VWR</td>
			<td>613-0798</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL disposable plastic cuvette</td>
			<td>Thomas Scientific</td>
			<td>8495V01</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>Sigma Aldrich</td>
			<td>D7039</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm Petri dish</td>
			<td>VWR</td>
			<td>391-1998</td>
			<td></td>
		</tr>
		<tr>
			<td>70% ethanol</td>
			<td>Solveco</td>
			<td>1054</td>
			<td></td>
		</tr>
		<tr>
			<td>Cdk1/2 inhibitor III</td>
			<td>217714</td>
			<td>Calbiochem</td>
			<td>300nM in 0.01% DMSO</td>
		</tr>
		<tr>
			<td>Cell culture incubator</td>
			<td>Thermo Forma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Life Technologies</td>
			<td>41966-029</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrodes</td>
			<td></td>
			<td></td>
			<td>Platina, custom made</td>
		</tr>
		<tr>
			<td>Electro square porator ECM 830</td>
			<td>Harvard Apparatus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>F12 Nutrient mix</td>
			<td>Life Technologies</td>
			<td>31331-028</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Life Technologies</td>
			<td>16140-071</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>AgnThos</td>
			<td>0108-5-PS</td>
			<td></td>
		</tr>
		<tr>
			<td>Freezing medium NEG50</td>
			<td>Cellab, Sweden</td>
			<td>6502</td>
			<td></td>
		</tr>
		<tr>
			<td>GFP expressing DNA plasmid (pZGs)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Humidified incubator</td>
			<td>Grumbach Brutgeraete GmbH, Asslar, Germany</td>
			<td>8204</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma Aldrich</td>
			<td>I9278-5ML</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Life Technologies</td>
			<td>25030024</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting medium ProLong Gold with DAPI</td>
			<td>Life Technologies</td>
			<td>P36935</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin film</td>
			<td>VWR</td>
			<td>291-1214P</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma Aldrich</td>
			<td>16005-1KG-R</td>
			<td></td>
		</tr>
		<tr>
			<td>Peel-A-Way embedding mold&#160;</td>
			<td>Sigma Aldrich</td>
			<td>E6032</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin streptomycin</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>PhosphoHistone 3 (PH3)&#160;</td>
			<td>Millipore</td>
			<td>06-570</td>
			<td>Dilution 1/4000</td>
		</tr>
		<tr>
			<td>Platinum electrodes (custom made from &#34;rondelles&#34;)</td>
			<td>Sargenta</td>
			<td>390-R (rondeller)</td>
			<td>Dia: 4mm, 0.1 mm thickness</td>
		</tr>
		<tr>
			<td>Platimun electrodes&#160;</td>
			<td>Sonidel</td>
			<td>CUY700P4L</td>
			<td>Dia: 4 mm</td>
		</tr>
		<tr>
			<td>Polyethylene pasteur pipette</td>
			<td>VWR</td>
			<td>612-2853</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotator shaker</td>
			<td>VWR</td>
			<td>444-2900</td>
			<td></td>
		</tr>
		<tr>
			<td>Small spoon</td>
			<td>VWR</td>
			<td>231-2151</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>VWR</td>
			<td>443815S</td>
			<td></td>
		</tr>
		<tr>
			<td>White Leghorn eggs</td>
			<td></td>
			<td></td>
			<td>Local supplier</td>
		</tr>
		<tr>
			<td>Wine cooler</td>
			<td>WineMaster 24, Caso, Berlin Germany</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

whole retinal explants from chicken embryos for electroporation and chemical reagent treatments

The retina is a good model for the developing central nervous system. The large size of the eye and most importantly the accessibility for experimental manipulations in ovo/in vivo makes the chicken embryonic retina a versatile and very efficient experimental model. Although the chicken retina is easy to target in ovo by intraocular injections or electroporation, the effective and exact concentration of the reagents within the retina may be difficult to fully control. This may be due to variations of the exact injection site, leakage from the eye or uneven diffusion of the substances. Furthermore, the frequency of malformations and mortality after invasive manipulations such as electroporation is rather high.
This protocol describes an ex ovo technique for culturing whole retinal explants from chicken embryos and provides a method for controlled exposure of the retina to reagents. The protocol describes how to dissect, experimentally manipulate, and culture whole retinal explants from chicken embryos. The explants can be cultured for approximately 24 hr and be subjected to different manipulations such as electroporation. The major advantages are that the experiment is not dependent on the survival of the embryo and that the concentration of the introduced reagent can be varied and controlled in order to determine and optimize the effective concentration. Furthermore, the technique is rapid, cheap and together with its high experimental success rate, it ensures reproducible results. It should be emphasized that it serves as an excellent complement to experiments performed in ovo.

This protocol describes the preparation (Figure 1A-F) and culturing of whole retinal explants from chicken embryos. This protocol has been successfully used for whole retinal explants from embryos of st20 (E3) to st31 (E7).
Electroporation of DNA plasmids into whole retinal explants allows for labeling and tracing of retinal progenitor cells or over-expression of different gene products. For electroporation experiments, the pigment epithelium was carefully removed from the enu...

Watch this Scientific Journal Video about Whole Retinal Explants from Chicken Embryos for Electroporation and Chemical Reagent Treatments at JoVE.com

Whole Retinal Explants from Chicken Embryos for Electroporation and Chemical Reagent Treatments

This protocol describes a method to dissect, experimentally manipulate and culture whole retinal explants from chicken embryos. The explant cultures are useful when high success rate, efficacy and reproducibility are needed to test the effects of plasmids for electroporation and/or reagent substances, i.e., enzymatic inhibitors.

The retina is a good model for the developing central nervous system. The large size of the eye and most importantly the accessibility for experimental ...

whole-retinal-explants-from-chicken-embryos-for-electroporation

Research

JoVE Journal

Developmental Biology

9.4K vistas.  Uppsala University.  Este protocolo describe un método para diseccionar, experimentalmente manipular y explantes de cultivo completo de la retina a partir de embriones de pollo. Los cultivos de explantes son útiles cuando alta tasa de éxito, la eficacia y reproducibilidad son necesarios para probar los efectos de plásmidos para la electroporación y / o sustancias de reactivos, es decir, inhibidores enzimáticos. 