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 increases mortality. Extended storage time increases both mortality and abnormal development.</li>
	<li>Incubate the eggs in a 37.5 &#176;C and 60% humidified egg incubator with gentle rocking at a frequency of 5 times per 24 hr.</li>
	<li>Remove the incubated eggs from the egg incubator at the desired embryonic age. 
	Note: The age of the chicken embryo is determined as the time of incubation and is denoted as embryonic days (E) or as stages (st), according to the series of developmental stages described by Hamburger and Hamilton12.</li>
	<li>Place the selected eggs in the laminar flow hood. Wipe the eggshell with 70% ethanol to avoid contamination of the embryo. 
	Note: Conduct all procedures under aseptic conditions with sterile&#160;solutions&#160;<url>https://www-jove-com.remotexs.ntu.edu.sg/science-education/5030/making-solutions-in-the-laboratory&#34;lutions </url> and instruments.</li>
	<li>Tap the side of the egg firmly against a hard surface to crack open the egg. Place the content in a 100 mm Petri dish. Use a small spoon to transfer the embryo to a 35 mm Petri dish containing pre-warmed (37 &#176;C) 1x PBS to prevent the tissue from drying.</li>
	<li>Place the 35 mm Petri dish containing the embryonic tissue onto a binocular stereovision dissecting microscope in the laminar flow hood.&#160;Decapitate the embryo by pinching the neck with fine forceps and discard the body.</li>
	<li>Use fine forceps to make an incision through the mouth, ventral to the eye. Starting at the site of incision, tear open the tissue surrounding the entire eye. Pinch off the optic nerve and remove the intact eye from the eye socket. Collect both the left and right eye from the same embryo for use as either the control or the treated eye.</li>
	<li>Use a small spoon to transfer the eyes to a new 35 mm Petri dish containing pre-warmed (37 &#176;C) 1x PBS.</li>
</ol>
2. Preparation of Whole Retinal Explants
<ol>
	<li>Use fine forceps to remove all tissue around the eye including the scleral anlage. 
	Note: The tissue will detach easily leaving the retina on the vitreous body and lens with the pigment epithelium intact (Figure 1A-B).</li>
	<li>Use fine forceps to pinch a small incision into the pigment epithelium at the dorsal part of the eye without damaging the neural retina (Figure 1C).</li>
	<li>Use fine forceps to gently tear open the pigment epithelium starting at the site of incision, leaving the lens and the entire retina attached to the vitreous body (Figure 1D). Keep the eye in warm (37 &#176;C) 1x PBS to facilitate the removal of the pigment epithelium. 
	Note: The pigment epithelium may be difficult to remove, in particular along the ciliary body around the pupil, in the anterior region of the eye, and along the choroid fissure. Therefore, some of the pigment epithelium may be left on the neural retina (Figure 1E-F).</li>
</ol>
3. Treatment of Whole Retinal Explants
<ol start="1">
	<li>Electroporation of whole retinal explants 
	Note: Electroporation of whole retinal explants can be performed directly after step 2.
	<ol>
		<li>Prepare culture medium containing 1:1 DMEM:F12 Nutrient mix, 10% FBS, 10 U/ml&#160;penicillin streptomycin, 5 &#181;g/ml&#160;Insulin, and 2 mM L-glutamine.</li>
		<li>Cut off a 1.5 ml&#160;disposable plastic cuvette (12.5 mm x 12.5 mm x 45 mm) (or any small container capable of holding 300-400 &#181;l&#160;solution) approximately 8 mm from the bottom using a fine-bladed saw.</li>
		<li>Dilute the DNA plasmid of choice in 1xDPBS to a final concentration of 0.1 &#181;g/&#181;l.</li>
		<li>Turn on the electroporator and set parameters to 15 V&#160;for st20 &#8211; st25 (E3-E4) retina and 20 V&#160;for st26 &#8211; st27 (E5) retina. Set the pulse-repeats to 5 pulses of 50 msec pulse-length with 1 sec intervals. Use a pulse generator that can be controlled by a foot-pedal on the floor as both hands will be needed to hold the electrodes in place.</li>
		<li>Connect two paddle shaped (flat, circular diam. 4 mm) platinum electrodes (Figure 2A) to the output socket on the pulse generator. 
		Note: The platinum electrodes used in this protocol were made by the university workshop. The electrodes were made from 0.1 mm platinum plate &#8220;rondelles&#8221; (platinum grade: 950 Pt/5 Cu). The connecting 0.8 mm wire was insulated using plastic tubing.</li>
		<li>Carefully transfer the whole retinal explant from step 2.3 to the cuvette (or equivalent small container) using a polyethylene Pasteur pipette with the tip cut off to acquire the right tip-opening size for the retinal explant. Avoid using pipette tips as the retina tends to attach to the plastic and tear apart.</li>
		<li>Use a 100 &#181;l pipette to gently remove the entire 1x PBS surrounding the retinal explant taking care not to touch the retina to avoid tearing.</li>
		<li>Add 100 &#181;l plasmid solution to the cuvette to cover the whole retinal explant. Gently push down the retinal explant with forceps if it floats to the top. Use forceps or electrodes to position the lens to face any one side of the cuvette and the optic nerve to the bottom of the cuvette.</li>
		<li>Place the positive electrode in front of the lens and the negative electrode behind the retina (Figure 2A), without touching the tissue.</li>
		<li>Apply the current by pressing down the foot-pedal. Check for bubbles at the electrodes indicating successful discharge.</li>
		<li>Use a 100 &#181;l pipette to remove all the plasmid mix from the cuvette without damaging the retina. The plasmid mix can be reused three to five times.</li>
		<li>Fill up the cuvette with pre-warmed (37 &#176;C) 1x PBS. Use forceps to gently detach the retinal explant from the wall of the cuvette. After electroporation the retina is sometimes covered with bubbles, which makes it adhere to the wall of the cuvette.</li>
		<li>Use the polyethylene Pasteur pipette to transfer the whole retinal explant into a 24-well plate containing 1 ml&#160;pre-warmed (37 &#176;C) culture medium per well. Incubate one retinal explant per well.</li>
		<li>Incubate the retinal explant at 37 &#176;C on a rotator shaker, with a constant speed of 50 rpm, inside a cell incubator with 5% CO2 for 24 hr. The rotation is to ensure maximum exposure to the medium and to prevent adhesion of the retina to the bottom of the 24-well plate.</li>
		<li>Proceed to step 4.</li>
	</ol>
	</li>
	<li>Treatment of whole retinal explants with chemical reagents 
	Note: Chemical treatment of whole retinal explants can be performed directly after step 2.
	<ol>
		<li>Prepare culture medium containing 1:1 DMEM:F12 Nutrient mix, 10% FBS, 10 U/ml&#160;penicillin streptomycin, 5 &#181;g/ml&#160;Insulin, and 2 mM L-glutamine.</li>
		<li>Use a small spoon to transfer the retinal explant from step 2.3 into a 24-well plate containing 1 ml&#160;pre-warmed (37 &#176;C) culture medium per well. Incubate one retinal explant per well.</li>
		<li>Incubate the whole retinal explant for 60 min at 37 &#176;C on a rotator shaker, with a constant speed of 50 rpm, inside a cell incubator with 5% CO2 before adding a chemical reagent or vehicle.</li>
		<li>Prepare the chemical solution, for example Cdk1/2 inhibitor III, an inhibitor of M-phase progression13, at a final concentration of 30 &#181;M in DMSO.</li>
		<li>Remove the 24-well plate, containing the whole retinal explant, from the cell incubator. Add 10 &#181;l of the chemical reagent or the vehicle (DMSO) directly to the retinal medium, resulting in a final concentration of 300 nM Cdk1/2 inhibitor III in 0.01% DMSO. Manually rotate the 24-well plate to allow for even distribution of the chemical reagent or vehicle in the solution.</li>
		<li>Incubate the retinal explants at 37 &#176;C on a rotator shaker, with a constant speed of 50 rpm, inside an incubator with 5% CO2 for the desired time (up to 24 hr).</li>
		<li>Proceed to step 4.</li>
	</ol>
	</li>
</ol>
4. Fixation and Freezing of Whole Retinal Eexplants
<ol>
	<li>Remove the 24-well plate containing the treated whole retinal explant from the cell incubator.</li>
	<li>Use a small spoon to transfer the retinal explant to a 24-well plate containing 1 ml/well ice cold 4% paraformaldehyde in 1xPBS. Incubate on ice for 15 min.</li>
	<li>Use a small spoon to transfer the whole retinal explant to a 24-well plate containing 1 ml/well ice cold 1xPBS to wash the retina. Incubate on ice for 10 min.</li>
	<li>Use a small spoon to transfer the whole retinal explant to a 24-well plate containing 1 ml/well ice cold 30% sucrose in 1xPBS. Incubate on ice for 1-3 hr&#160;depending on the embryonic day of the retina. For st20 &#8211; st25 (E3) retinas, incubate for 1 hr, older retinas need longer incubation time in sucrose.</li>
	<li>Use a small spoon to transfer the whole retinal explant onto a paraffin film containing approximately 500 &#181;l&#160;cryostat freezing mounting medium. Remove as much sucrose as possible by gently moving the retinal explant in the freezing medium using the small spoon.</li>
	<li>Transfer the retinal explant to an embedding mold containing freezing medium. Position the eye to facilitate future sectioning. Put the embedding mold containing the whole retinal explant on dry ice until the medium is frozen solid. Store at -80 &#176;C.</li>
</ol>

<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. 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The authors declare that they have no competing financial interests.

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 defined DNA plasmid concentration. This approach&#160;allows for a reliable method to investigate gene function in the retina with a typically higher success rate compared to electroporations in ovo. The whole retinal explant also makes it possible to expose the retina to a medium containing a defined concentration of a chemical reagent giving a uniform distribution of the reagent in the neural retina. The protocol allows for combined treatments where the retina is first electroporated and then treated with substances. The influence of variability between individual embryos may be minimized, since both the treated and control eye can be collected from the same embryo and be treated independently.
The protocol has been optimized for whole retinal explants from st20 (E3) to st27 (E5) embryos for electroporation and st20 (E3) to st31 (E7) embryos for chemical substance treatment. At younger stages the retina/optic cup is too small and fragile to effectively handle and the in ovo electroporation is most likely more efficient. Electroporations on whole retinal explants was not performed on stages older than st27 (E5). It is likely possible to adapt this protocol to explants from older embryos. That would include obtaining larger cuvettes, larger wells for incubation and increased volume of retinal culture medium. However, by E12-14 the pigment epithelium and the outer segment of the photoreceptor cells become closely associated, and removal of the pigment epithelium may damage the retina. The retina should be intact during the entire experimental procedure and incubation.
With this dissection and culturing protocol, the structural integrity of the retinal architecture remains intact during 24 hr, based on the expression of cell type specific markers. As mentioned, we have studied cell cycle progression in the explants and the process of interkinetic nuclear migration of retinal progenitor cells looks normal. If the protocol is performed swiftly there are only small effects on the developmental progression in the explant. However, the development may be slightly decelerated and an explant that has been cultured for 24 hr may exhibit a developmental age that is 1-2 hr younger than the normal counterpart. It is advised to compare the explant to age-matched normal retina and it is advised to use the contralateral eye as an experimental explant control. It should be pointed out that removal of the retinal pigment epithelium may affect the development of the outer photoreceptor segments. It should also be pointed out that an explant retina represents an injured retina with axotomized retinal ganglion cells.
This protocol is simple and therefore reduces experimental errors but the ex ovo situation demands several experimental controls. A specific developmental process that is subject to be studied using this ex ovo protocol may also be studied in parallel and be compared to the normal in ovo situation. The appropriate experimental controls must always be included, such as vehicles used to solubilize substances. When using signal pathway blockers, use several different inhibitors for the same process or use different inhibitors that target enzymes at more than one level of the specific pathway that is studied. For electroporation, a GFP-expressing DNA plasmid is useful as a positive control. If such plasmid does not produce any reporter gene expression, it is most likely due to that the anode (+) and cathode (-) are incorrectly positioned. The DNA plasmid will then migrate away from the retina. When testing novel DNA plasmids it is therefore recommended to include a DNA plasmid that expresses a different fluorescent protein as an internal electroporation control. The electroporation parameters described in this protocol have been optimized to produce a high number of reporter gene expressing cells. Decreasing the voltage or the number of pulses reduces the number of transfected cells, which may produce false negative results. However, increasing the voltage or the number of pulses may lead to damage of the tissue and increased cell death. Three sets of custom made platinum electrodes have been used for all electroporations during more than two years without showing any reduced capacity. The electrodes must be carefully cleaned and the electrode surface be polished. Commercial 4 mm paddle electrodes are available.
This protocol focuses on electroporation of DNA plasmids and chemical treatment of whole retinal explants. It is possible to further expand this protocol to include other experimental procedures, such as a knock-down of gene expression with the use of morpholino oligomers. The whole retinal explant procedure opens up for the possibility to perform short-term experiments in an easy, quick and highly reproducible way. It reduces costs, time, and the number of animals needed for each experiment and most importantly it increases the possibility to get solid data.

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><tbody><tr><td>1xPBS (tablet)</td><td>Life technologies</td><td>18912-014</td><td></td></tr><tr><td>10x DPBS</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 &amp;mu;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>300 nM 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&amp;nbsp;</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)&amp;nbsp;</td><td>Millipore</td><td>06-570</td><td>Dilution 1/4,000</td></tr><tr><td>Platinum electrodes (custom made from &quot;rondelles&quot;)</td><td>Sargenta</td><td>390-R (rondeller)</td><td>Dia: 4mm, 0.1 mm thickness</td></tr><tr><td>Platimun electrodes&amp;nbsp;</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 enucleated eye of a st25 (E4 &#189;) embryo. The whole retinal explant was then placed in a cuvette containing the DNA plasmid solution, electrodes were submerged (Figure 2A) and a current was applied. After 24 hr of culturing, GFP positive cells were visible in a large part of the intact retina (Figure 2B). After sectioning, single GFP+ cells were easily detected along the apico-basal axis of the retina (Figure 2C). Electroporation resulted in a large retinal area taking up the plasmid and expressing the reporter gene. The success rate, measured by the number of retinas with an electroporated area larger than 1/5 of the total retina, was 75% (62 out of 83 retinas) for whole retinal explant electroporation, compared to 14% (37 out of 263 eyes) for in ovo electroporation. Electroporation experiments on whole retinal explants have successfully been performed on st20 (E3) to st27 (E5) embryos.
The whole retinal explants can be cultured for approximately 24 hr (Figure 3A and B). Longer incubation times may lead to developmental delay, morphological malformations, apoptosis and eventually disintegration of the retina (Figure 3C). When culturing the chicken retinas as whole explants the architecture of the retina remains intact. This includes distribution of the different phases of the cell cycle along the apico-basal axis. During normal development the cells undergo S-phase on the basal side, followed by mitosis on the apical side of the retina14,15. A section from a whole retinal explant, cultured for 24 hr, was immunostained with a Phospho-Histone 3 (PH3) antibody, labeling cells in late G2/M-phase (Figure 3D). The PH3 positive cells were found on the apical side of the retina, consistent with normal retinal development. An active cyclin B1-Cdk1 complex in the nucleus will initiate M-phase transition13. Blocking the Cdk1-kinase activity will inhibit down-stream events that are necessary for cell cycle propagation into mitosis. Stage 29 (E6) whole retinal explants were incubated with the Cdk1/2 Inhibitor III for 4 hr and PH3 immunoreactivity was analyzed. The Cdk1/2 inhibitor III reduced the number of PH3 positive cells (Figure 3E), indicating an efficient block of G2/M-phase transition. The reduction of the number of mitoses after treatment with the Cdk1/2 inhibitor verified that the treatment was effective.
<img alt="Figure 1" src="/files/ftp_upload/53202/53202fig1.jpg" /> 
Figure 1. Removal of the pigment epithelium. An embryonic day 6 (st29) chicken eye was dissected and prepared for culturing as whole retinal explant. (A) View of the anterior eye with pupil and lens and the choroid fissure facing down. The pigment epithelium is intact but the scleral anlage and surrounding connective tissue has been removed. (B) View of the posterior part of the eye. The optic nerve exit and choroid fissure are facing down. (C) Incision in the pigment epithelium. (D) Most of the pigment epithelium has been removed. (E) Some pigment epithelium is left at along the rim of the ciliary body and (F) the choroid fissure. LE: lens and ciliary body, CF: choroid fissure, ON: optic nerve exit. Scale bar is 0.5 mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53202/53202fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53202/53202fig2.jpg" /> 
Figure 2. Electroporation of whole retinal explants. (A) Schematic presentation of the platinum electrodes and how they are positioned on either side of the whole retinal explant that is submerged in DNA plasmid solution in the truncated cuvette. (B) Whole retinal explant after 24 hr&#160;of culturing, and (C) sectioned retina. ON: optic nerve exit. Scale bar is (A) 4 mm, (B) 0.5 mm, (C) 50 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53202/53202fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53202/53202fig3.jpg" /> 
Figure 3. Cultured whole retinal explants were fixed, frozen, cryosectioned, and labeled by immunohistochemistry. (A) A st29 whole retinal explant cultured for 24 hr. Fluorescence micrographs of nuclear staining after (B) 24 hr&#160;and (C) 48 hr. (D) A PH3 antibody was used to label cells in late G2/M-phase. (E) The relative density (PH3+ cells/mm2, mean &#177; SD) and fluorescence micrographs of PH3+ cells at st29 after Cdk1/2 inhibitor III treatment for 4 hr&#160;compared to vehicle. st: Hamburger and Hamilton stages, Student&#8217;s t-test, ** p &#60; 0.01, n &#8805; 4 treated eyes, 4 sections per eye, h: hours. Scale bar is (A) 0.5 mm, (B-E) 10 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53202/53202fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

9.4K Views.  Uppsala University.  Dit protocol beschrijft een methode te ontleden, experimenteel manipuleren en cultuur hele netvlies explantaten uit kippenembryo's. De explantkweken zijn bruikbaar wanneer hoge succespercentage, werkzaamheid en reproduceerbaarheid nodig om de effecten van elektroporatie van plasmiden en / of reagens stoffen, dat wil zeggen enzymatische inhibitoren testen. 

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

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying the biology of retinal neurons, particularly photoreceptors. The applications of this system go beyond basic research, as they can easily be adapted to high throughput technologies for drug development. However, genetic manipulation of retinal photoreceptors in these cultures has proven to be very challenging, posing an important limitation to the usefulness of the system. We have recently developed and validated an ex&#160;ovo plasmid electroporation technique that increases the rate of transfection of retinal cells in these cultures by five-fold compared to other currently available protocols1. In this method embryonic chick eyes are enucleated at stage 27, the RPE is removed, and the retinal cup is placed in a plasmid-containing solution and electroporated using easily constructed custom-made electrodes. The retinas are then dissociated and cultured using standard procedures. This technique can be applied to overexpression studies as well as to the downregulation of gene expression, for example via the use of plasmid-driven RNAi technology, commonly achieving transgene expression in 25% of the photoreceptor population. The video format of the present publication will make this technology easily accessible to researchers in the field, enabling the study of gene function in primary retinal cultures. We have also included detailed explanations of the critical steps of this procedure for a successful outcome and reproducibility.

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

Embryonic chick retinal cell cultures constitute valuable tools for the study of photoreceptor biology. We have developed an efficient gene transfer technique based on ex&#160;ovo plasmid electroporation of the retinas prior to culture. This technique considerably increases transfection efficiencies over currently available protocols, making genetic manipulation feasible for this system.

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying ...

In Ovo Electroporations of HH Stage 10 Chicken Embryos

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology.  With the advent of molecular biological techniques, the chick has become a useful system in which to study gene regulation and function.  By electroporating DNA or RNA constructs into the developing chicken embryo, genes can be expressed or knocked down in order to analyze in vivo gene function.  Similarly, reporter constructs can be used for fate mapping or to examine putative gene regulatory elements.  Compared to similar experiments in mouse, chick electroporation has the advantages of being quick, easy and inexpensive.  This video demonstrates first how to make a window in the eggshell to manipulate the embryo.  Next, the embryo is visualized with a dilute solution of India ink injected below the embryo.  A glass needle and pipette are used to inject DNA and Fast Green dye into the developing neural tube, then platinum electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator.  Finally, the egg is sealed with tape and placed back into an incubator for further development.  Additionally, the video shows proper egg storage and handling and discusses possible causes of embryo loss following electroporation.

Chick in ovo electroporation is a technique which allows genetic manipulation of the avian embryo. Common applications of this technique include functional analysis of genes and putative enhancer elements. This video demonstrates neural tube electroporation in HH 10 chick embryos. Injection technique and proper egg handling are discussed.

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology.  With the advent of ...

Biology

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Neuroscience

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.

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.

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

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell differentiation, axon guidance, retinotopic organization and synapse formation[1]. One drawback is the inability to efficiently and reliably manipulate gene expression in RGCs in vivo, especially in the otherwise accessible murine visual pathways. Transgenic mice can be used to manipulate gene expression, but this approach is often expensive, time consuming, and can produce unwanted side effects. In chick, in ovo electroporation is used to manipulate gene expression in RGCs for examining retina and RGC development. Although similar electroporation techniques have been developed in neonatal mouse pups[2], adult rats[3], and embryonic murine retinae in vitro[4], none of these strategies allow full characterization of RGC development and axon projections in vivo. To this end, we have developed two applications of electroporation, one in utero and the other ex vivo, to specifically target embryonic murine RGCs[5, 6]. 
With in utero retinal electroporation, we can misexpress or downregulate specific genes in RGCs and follow their axon projections through the visual pathways in vivo, allowing examination of guidance decisions at intermediate targets, such as the optic chiasm, or at target regions, such as the lateral geniculate nucleus. Perturbing gene expression in a subset of RGCs in an otherwise wild-type background facilitates an understanding of gene function throughout the retinal pathway. Additionally, we have developed a companion technique for analyzing RGC axon growth in vitro. We electroporate embryonic heads ex vivo, collect and incubate the whole retina, then prepare explants from these retinae several days later. Retinal explants can be used in a variety of in vitro assays in order to examine the response of electroporated RGC axons to guidance cues or other factors. In sum, this set of techniques enhances our ability to misexpress or downregulate genes in RGCs and should greatly aid studies examining RGC development and axon projections.

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

Here we present two techniques for manipulating gene expression in murine retinal ganglion cells (RGCs) by in utero and ex vivo electroporation. These techniques enable one to examine how alterations in gene expression affect RGC development, axon guidance, and functional properties.

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell ...

Visualization of Tangential Cell Migration in the Developing Chick Optic Tectum

Time-lapse imaging is a powerful method to analyze migrating cell behavior. After fluorescent cell labeling, the movement of the labeled cells in culture can be recorded under video microscopy. For analyzing cell migration in the developing brain, slice culture is commonly used to observe cell migration parallel to the slice section, such as radial cell migration. However, limited information can be obtained from the slice culture method to analyze cell migration perpendicular to the slice section, such as tangential cell migration. Here, we present the protocols for time-lapse imaging to visualize tangential cell migration in the developing chick optic tectum. A combination of cell labeling by electroporation in ovo and a subsequent flat-mount culture on the cell culture insert enables detection of migrating cell movement in the horizontal plane. Moreover, our method facilitates detection of both individual cell behavior and the collective action of a group of cells in the long term. This method can potentially be applied to detect the sequential change of the fluorescent-labeled micro-structure, including the axonal elongation in the neural tissue or cell displacement in the non-neural tissue.

We describe the methods for fluorescent labeling of tangentially migrating cells by electroporation, and for time-lapse imaging of the labeled cell movement in a flat-mount culture in order to visualize migrating cell behavior in the developing chick optic tectum.

Time-lapse imaging is a powerful method to analyze migrating cell behavior. After fluorescent cell labeling, the movement of the labeled cells in culture ...

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.

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.

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

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

The chicken embryo provides an excellent model system for studying gene function and regulation during embryonic development. In ovo electroporation is a powerful method to over-express exogenous genes or down-regulate endogenous genes in vivo in chicken embryos1. Different structures such as DNA plasmids encoding genes2-4, small interfering RNA (siRNA) plasmids5, small synthetic RNA oligos6, and morpholino antisense oligonucleotides7 can be easily transfected into chicken embryos by electroporation. However, the application of in ovo electroporation is limited to embryos at early incubation stages (younger than stage HH20 - according to Hamburg and Hamilton)8 and there are some disadvantages for its application in embryos at later stages (older than stage HH22 - approximately 3.5 days of development). For example, the vitelline membrane at later stages is usually stuck to the shall membrane and opening a window in the shell causes rupture of the vessels, resulting in death of the embryos; older embryos are covered by vitelline and allantoic vessels, where it is difficult to access and manipulate the embryos; older embryos move vigorously and is difficult to control the orientation through a relatively small window in the shell.
In this protocol we demonstrate an ex ovo electroporation method for gene transfer into chicken embryos at late stages (older than stage HH22). For ex ovo electroporation, embryos are cultured in Petri dishes9 and the vitelline and allantoic vessels are widely spread. Under these conditions, the older chicken embryos are easily accessed and manipulated. Therefore, this method overcomes the disadvantages of in ovo electroporation applied to the older chicken embryos. Using this method, plasmids can be easily transfected into different parts of the older chicken embryos10-12.

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

A method of gene transfer into chicken embryos at later incubation stages (older than Hamburger and Hamilton stage (HH) 22) is described. This method overcomes disadvantages of in ovo electroporation applied to older chicken embryos and is a useful technique to study gene function and regulation at older developmental stages.

The chicken embryo provides an  excellent model system for studying gene function and regulation during  embryonic development. In ovo electroporation is ...

Cone-Enriched Cultures from the Retina of Chicken Embryos to Study Rod to Cone Cellular Interactions

Human daytime vision relies on the function of cone photoreceptors at the center of the retina, the fovea. Patients suffering from the most prevalent form of inherited retinal degeneration, retinitis pigmentosa, lose night vision because of mutation driven loss of rod photoreceptors, a phenomenon followed by a progressive loss of function and death of cones leading to blindness. Geneticists have identified many genes with mutations causing this disease, but the first mutations identified questioned the mechanisms of secondary cone degeneration and how a dominant mutation in the rhodopsin gene encoding for the visual pigment expressed exclusively in rods can trigger cone degeneration.
This result of transplantations in a genetic model of the disease led to the concept of cell interactions between rods and cones and of non-cell autonomous degeneration of cones in all genetic forms of retinitis pigmentosa.
Cones comprise 5% of all photoreceptors in humans and only 3% in the mouse, so their study is difficult in these species, but cones outnumber rods in bird species. We have adapted 96-well plates to culture retinal precursors from the retina of chicken embryos at stage 29 of their development. In these primary cultures, cones represent 80% of the cells after in vitro differentiation. The cells degenerate over a period of one week in the absence of serum. Here, we describe the methods and its standardization.
This cone-enriched culture system was used to identify the epithelium-derived cone viability factor (EdCVF) by high content screening of a rat retinal pigmented epithelium normalized cDNA library. Recombinant EdCVF prevents the degeneration of the cones.

We describe a method to obtain primary cultures of cone photoreceptors from the retina of chicken embryos and its use for high content screening.

Human daytime vision relies on the function of cone photoreceptors at the center of the retina, the fovea. Patients suffering from the most prevalent form ...

Loss-of-Function Approach in the Embryonic Chick Retina by Using Tol2 Transposon-Mediated Transgenic Expression of Artificial microRNAs

The chick retina has long been an important model system in developmental neurobiology, with advantages including its large size, rapid development, and accessibility for visualization and experimental manipulations. However, its major technical limitation had been the lack of robust loss-of-function approaches for gene function analyses. This protocol describes a methodology of gene silencing in the developing chick retina that involves transgenic expression of artificial microRNAs (miRNAs) by using the Tol2 transposon system. In this approach, a Tol2 transposon plasmid that contains an expression cassette for the EmGFP (emerald green fluorescent protein) marker and artificial pre-miRNA sequences against a target gene is introduced into the embryonic chick retina with a Tol2 transposase expression construct by in ovo electroporation. In the transfected retinal cells, the transposase catalyzes the excision of the expression cassette from the transposon vector and its integration into host chromosomes, leading to the stable expression of miRNAs and the EmGFP protein. In our previous study, we have demonstrated that the expression of Nel, a glycoprotein that exerts multiple functions in neural development, can be significantly suppressed in the developing chick retina by using this technique. Our results indicate that this methodology induces a stable and robust suppression of gene expression and thus provides an efficient loss-of-function approach for studies of retinal development.

We have developed a novel loss-of-function approach that involves the introduction and genomic integration of artificial micro-RNA sequences into chick embryos by using in ovo electroporation and the Tol2 transposon system. This technique provides a robust and stable gene knockdown methodology for studies of gene function during development.

The chick retina has long been an important model system in developmental neurobiology, with advantages including its large size, rapid development, and ...

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

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