The pT2K-CAGGS and pCAGGS-T2TP vectors were kindly provided by Yoshiko Takahashi (Kyoto University, Kyoto, Japan) and Koichi Kawakami (National Institute of Genetics, Mishima, Japan), respectively. We thank Michael Berberoglu for his crucial reading of the manuscript. This work was supported by grants from the Royal Society and Biotechnology and Biological Sciences Research Council (BBSRC) (UK) to M.N.

1. Construction of miRNA expression vectors
NOTE: The procedures for constructing miRNA expression vectors (steps 1.1-1.3, 1.5-1.6.) are optimized for the miRNA expression kit, Block-iT Pol II miR RNA expression kit with EmGFP, as previously described15,16. The kit provides the expression vector designed to allow miRNA expression (pcDNA6.2-GW/EmGFP-miRNA), a control vector (pcDNA6.2-GW/EmGFP-miRNA-negative control plasmid), accessory reagents, and instructions to produce miRNA expression vectors (see Table of Materials)17.
<ol>
	<li>Designing single-stranded DNA oligos encoding pre-miRNAs against the target gene: Design single-stranded DNA oligos (&#34;top strand&#34; oligos (target pre-miRNAs) and &#34;bottom strand&#34; oligos (complements of top strand oligos)) using the online tool, RNAi Designer (see Table of Materials). See Figure 2 for the required features of the single-stranded oligos (Figure 2A) and examples of target sequences (Figure 2B). 
	NOTE: It is recommended that 5-10 pre-miRNA sequences be generated for a given target gene and screened for knockdown activities in vitro (step 1.4).</li>
	<li>Annealing of the top- and bottom-strand oligos to generate a double-stranded oligo
	<ol>
		<li>Set up the following annealing reaction (Table 1) in a sterile 0.5 mL&#160;microcentrifuge tube.</li>
		<li>Incubate the reaction mixture at 95 &#176;C for 4 min. Anneal the top- and bottom-strand oligos to generate a double-stranded oligo by allowing the reaction mixture to cool to room temperature (RT) for 5-10 min. Centrifuge the sample briefly (~5 s). 
		NOTE: The annealed oligos can be stored at -20 &#176;C without degradation for at least a year.</li>
	</ol>
	</li>
	<li>Cloning the double-stranded oligos into the miRNA expression vector (pcDNA6.2-GW/EmGFP-miRNA (Provided in the miRNA expression kit)): Clone individual double-stranded oligos into the linearized miRNA expression vector, according to the manufacturer&#39;s manual17.</li>
	<li>Evaluation of knockdown effects 
	NOTE: It is recommended that individual miRNA sequences be tested for gene suppression efficiency in vitro before they are applied in ovo. Knockdown efficiency can be tested by transfecting miRNA expression plasmids into a cell line that expresses the target gene. Alternatively, individual miRNA expression plasmids can be co-transfected into cell lines with an expression construct for the target gene. For target genes encoding proteins that are not membrane-anchored in their native state (e.g., soluble proteins), an alkaline phosphatase (AP) fusion protein can be used for monitoring the expression of the target protein. A cDNA sequence encoding the target protein can be fused in frame to human placental alkaline phosphatase in AP-tag vectors (APtag-1- APtag-5; see Table of Materials) and introduced into cells18. When expressed in culture cells (e.g., HEK293T cells), the AP-tagged target protein is secreted at high levels into culture media, and thus knockdown effects of miRNA sequences can be evaluated by measuring the decrease in AP activity in the culture media of miRNA-transfected cells (substeps 1.4.1-1.4.4).
	<ol>
		<li>Culture HEK293T cells in a 24-well plate (8 x 104 cells/well) overnight. Transiently transfect the cells with individual miRNA expression constructs with an expression plasmid of an AP-tagged target protein. Use the pcDNA6.2-GW/EmGFP-miRNA-negative control plasmid (provided in the miRNA expression kit) as a control. (If cell lines that stably express an AP-tagged target protein are used, transfect the cells only with individual miRNA expression constructs.) 
		NOTE: A conventional lipofection reagent (see Table of Materials) is used for transfection.</li>
		<li>Collect the conditioned medium 48-72 h after the transfection and heat inactivate the endogenous AP activity in a 65 &#176;C water bath for 5 min. Spinout debris in a desktop microcentrifuge at maximum speed for 5 min.</li>
		<li>Buffer the supernatant with 10 mM HEPES, pH 7.0 and pass it through a 0.45 &#181;m filter.</li>
		<li>Take 100 &#181;L (for measurement in a plate reader) or 500 &#181;L (for a spectrophotometer) of the supernatant and add an equal amount of 2x AP substrate buffer (Table 2). Check the AP activity by measuring OD405 in a plate reader or a spectrophotometer. 
		NOTE: If the AP activity of the conditioned medium is too high for accurate measurement, dilute it with HBAH buffer (Hanks&#39; balanced salt solution (HBSS), 0.5 mg/mL bovine serum albumin, 20 mM HEPES (pH 7.0)) or another buffer that contains a carrier protein. Do not use phosphate-containing buffers (e.g., PBS) because inorganic phosphate acts as a competitive inhibitor of AP.</li>
	</ol>
	</li>
	<li>Chaining of miRNA sequence 
	NOTE: Knockdown effects can be enhanced by chaining different miRNAs against the same target gene or repeating the same miRNA. The miRNA expression vector supports the chaining of multiple pre-miRNA sequences and their co-cistronic expression17.
	<ol>
		<li>Chain two different pre-miRNA sequences (against the same target gene) that show highest knockdown activities in in vitro assays (step 1.4), according to the manufacturer&#39;s instructions17.</li>
		<li>Evaluate gene suppression efficiency of the chained constructs using in vitro assays as described in step 1.4. If three or more pre-miRNA sequences show similarly high knockdown activities, test different combinations of two sequences and use the combinations that show the highest knockdown activity (see Representative Results).</li>
	</ol>
	</li>
	<li>Transferring the EmGFP-pre-miRNA expression cassette to the Tol2 transposon vector 
	NOTE: The expression cassette containing EmGFP cDNA and two pre-miRNA sequences is transferred into the Tol2 transposon vector (pT2K-CAGGS vector, see Table of Materials). To this end, the expression cassette (encompassing from the 3&#39; end of the CMV promoter to the miRNA reverse sequencing primer site of the miRNA expression vector) is PCR-amplified using primers with an artificial restriction enzyme site, and the PCR product is cloned into the Tol2 transposon vector. The Tol2 transposon vector contains the ubiquitous CAGGS promoter, which drives the expression of the inserted expression cassette. The CAGGS promoter and the expression cassette are flanked by the Tol2 sequences (Figure 1).
	<ol>
		<li>PCR amplification of the EmGFP-pre-miRNA expression cassette: Follow the reaction setup and the thermocycling conditions described in Table 3.</li>
		<li>Ligate the gel-purified PCR product (c. 1.3 kb) into the restriction enzyme-digested Tol2 transposon vector. Electroporate the plasmid into competent E. coli cells (see Table of Materials) and select the recombinants (pT2K-CAGGS-EmGFP-2x miRNA constructs).</li>
		<li>Prepare the plasmid by using a conventional maxiprep kit (see Table of Materials). Check the structure and sequence of the plasmid by restriction mapping and by using the primers used for the PCR, respectively.</li>
	</ol>
	</li>
</ol>
2. Egg storage and incubation
<ol>
	<li>Purchase fertilized White Leghorn (Gallus gallus) eggs from local farms or commercial vendors. 
	NOTE: Eggs may be kept at 12-16 &#176;C or at 4 &#176;C for up to 1 week prior to incubation without significant loss of viability or delay in development during incubation.</li>
	<li>Label the eggs with the start date of incubation and mark the top side of the egg (where the embryo will be positioned). Incubate fertilized eggs in a horizontal position at 38 &#176;C until the embryos have reached Hamburger and Hamilton (HH) stages 10 (33-38 h) -11 (40-45 h)19.</li>
</ol>
3. In ovo electroporation
<ol>
	<li>Preparation for in ovo electroporation
	<ol>
		<li>Preparation of 0.25% fast green solution: Dissolve 25 mg of Fast Green FCF in 10 mL of PBS. Filter the solution using a 0.2 &#181;m syringe filter. The solution can be stored at RT.</li>
		<li>DNA cocktail: Prepare the injection solution by mixing the individual pT2K-CAGGS-EmGFP-2x miRNA plasmids (substep 1.6.3) with the Tol2 transposase expression plasmid (pCAGGS-T2TP vector; see Table of Materials) (5 &#181;g/&#181;L each) at the ratio of 2:1. Add 1/10 volume of 0.25% fast green solution to visualize the injected area. 
		NOTE: The optimum DNA concentration may vary depending on the constructs.</li>
		<li>Setting up the microinjection apparatus (Figure 3A,B): The microinjection apparatus consists of the followings (see Table of Materials): Hamilton syringe, 18 G needle (Needle length = 2&#34;), PVC (Polyvinyl chloride) tubing (2 cm length), Micropipette needle (Can be made by pulling capillary tubes with omega dot fiber (1 mm O.D. X 0.75 mm I.D) with a micropipette puller).
		<ol>
			<li>Fill a Hamilton syringe with heavy mineral oil. Attach an 18 G needle to the syringe and fill the inner space of the needle with oil by depressing the syringe plunger.</li>
			<li>Attach a piece of 2 cm long PVC&#160;tubing to the end of the needle and fill the tubing with the oil.</li>
			<li>Attach a pulled micropipette needle to the tubing. Break off the tip of the micropipette needle to a 10-20 &#181;m diameter by fine forceps to make a small opening. Fill the entire needle with oil. 
			NOTE: Care should be taken not to trap any air bubbles in the system, as they would inhibit the flow of the DNA solution.</li>
		</ol>
		</li>
		<li>Put 5 &#181;L of the colored DNA cocktail (substep 3.1.2) onto a sterile Petri dish. Under the dissecting microscope, place the tip of the micropipette needle into the DNA solution on the sterile&#160;Petri dish and slowly draw the solution into the needle.</li>
		<li>Wait until the pressure equilibrates inside and outside the needle (to avoid that air goes into the needle) and take the tip of the needle out of the DNA solution. Keep the tip of the needle submerged in sterile PBS in a small beaker until injection.</li>
		<li>Setting up&#160;the electroporation apparatus (Figure 3A,C):&#160;
		<ol>
			<li>Set a pair of platinum electrodes&#160;with an electrode holder on a micromanipulator. Adjust the spacing between the tip of the electrodes to 2 mm (Figure 3C,E).</li>
			<li>Connect the electrodes to a square wave pulse generator with cables (see Table of Materials).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Microinjection of DNA solution (Figure 3D)
	<ol>
		<li>Remove a chicken egg from the incubator and wipe the surface of the egg with a tissue paper soaked in 70% ethanol.</li>
		<li>Attach an 18 G needle to a 10 mL syringe. Insert the needle through the blunt end of the egg, angled downward (45&#176;) to avoid damaging the yolk.</li>
		<li>Withdraw 2-3 mL of albumin from the egg. Seal the hole with a piece of Scotch tape. Confirm that the embryo and vitelline membrane are detached from the shell by &#34;candling&#34; the egg with light.</li>
		<li>Remove a piece of eggshell (2-3 cm diameter circle) from the top of the egg using scissors and forceps to expose the embryo. Do not window more than five eggs at any one time to prevent drying out of embryos during electroporation. 
		NOTE: If it is difficult to open a window without cracking the egg, the entire top of the egg can be covered with Sellotape or Scotch tape before removing a piece of eggshell.</li>
		<li>Insert the tip of the needle into the optic vesicle from its proximal side at a 45&#176; angle and inject the DNA cocktail by slowly depressing (or tapping on) the plunger until the blue-colored solution fills the lumen (Figure 3D). 
		NOTE: Alternatively, a pressure microinjection system (see Table of Materials) can be used for the injection of DNA solutions.</li>
		<li>Withdraw the needle and place its tip back into PBS.</li>
	</ol>
	</li>
	<li>Electroporation (Figure 3E)
	<ol>
		<li>Set pulse parameters of the electroporator as follows: Voltage: 15 V, Pulse length: 50 ms, Pulse interval: 950 ms, Pulse number: 5</li>
		<li>Add a few drops of HBSS onto the vitelline membrane over the embryo. Lower the electrodes into the HBSS using the micromanipulator, perpendicular to the anterior-posterior axis of the embryo. 
		NOTE: Alternatively, this procedure can be done without adding HBSS, as albumin is a good electric conductor that allows efficient electroporation.</li>
		<li>Place the electrodes on either side (anterior (nasal) side and posterior (temporal) side) of the optic vesicle (Figure 3E). Ensure that the electrodes do not touch the embryo or blood vessels. Apply pulsed electric fields.</li>
		<li>Remove the electrodes and gently clean the electrodes with a water-soaked sterile cotton bud to avoid the accumulation of albumin.</li>
		<li>Seal the window with Scotch tape and re-incubate the embryo until the desired developmental stage.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

This protocol provides a detailed guide to gene silencing in the developing chick retina by transgenic expression of artificial miRNAs using in ovo electroporation and the Tol2 transposon system.
The following factors are of critical importance in performing this technique successfully. First, it is critical to use miRNA sequences that are confirmed to exert robust knockdown effects. Before applying them for in ovo electroporation, test individual pre-miRNA sequences for gene suppression efficiency in in vitro assays (step 1.4) and chain two pre-miRNA sequences that show the highest knockdown activities (step 1.5). Second, it is important not to damage the optic vesicle and surrounding nervous tissues when inserting the micropipette needle for injection of the DNA solution. Excessive damage on embryos could lead to embryonic malformation and death. To avoid tissue damages, introduce the micropipette into the optic vesicle in the correct orientation. Smaller and sharper tips of the micropipette needle would make penetration of the optic vesicle smoother and result in less tissue damage. However, needles with very small tips have difficulty with loading and releasing the DNA solution. In the study described here, needles with a 10-20 &#181;m diameter tip opening were used. Third, make sure to fill the entire optic vesicle with the DNA solution (as visualized by the spread of fast green). Fourth, place the electrodes in the optimal positions to target DNAs into the desired area of the optic vesicle (e.g., temporal side, nasal side). Negatively charged DNA molecules will move towards the anode side; therefore the precise placement and orientation of the electrodes critically affect the final results. Finally, use the optimal DNA concentrations and electroporation parameters.
If no fluorescent signal is observed after 24 h of electroporation, the following should be considered: (1) Confirm that the electroporated plasmids have correct sequences. (2) Recheck the concentration and purity of individual plasmids. (3) Make sure that the DNA solution fills the lumen of the optic vesicle and it does not diffuse away into the neural tube. (4) Check the electroporation parameters used are correct. (5) Check that the electrodes are in contact with the vitelline membrane while the electric pulses are applied.
The combination of in ovo electroporation and a transposon-mediated gene integration method in this protocol offers several distinct advantages. First, it supports the stable production of miRNAs and thus persistent suppression of target genes. Development of the chick retina starts at E1.5 and continues until hatching (Retinal ganglion cells are produced over the period from E2 to E9). For loss-of-function analysis of gene function, expression of artificial miRNAs should be stably maintained during this period. Expression of RNAi sequences using conventional expression vectors is generally transient because the expression construct fails to be efficiently integrated into chromosomes. In the method described here, however, chromosomal integration of the expression cassette is mediated by the activity of co-electroporated transposase, which results in stable expression of the transgenes.
Second, in this method, the same CAGGS promoter induces co-cistronic expression of multiple miRNA sequences and the EmGFP marker in transfected cells, thus circumventing the risk of promoter interference, which is one of the common complications with retroviral vectors containing two promoters to attain dual gene expression25.
Finally, unlike replication-competent retroviral vectors, the transgene that is electroporated by this method is not transferred to neighboring cells. Therefore, the area of transfection can be more precisely controlled by using the appropriate types of electrodes and by placing them in the correct positions.
Despite the advantages described above, the current method also has limitations that are inherent to in ovo electroporation. For example, the rates of transfection and gene suppression may vary between embryos, and a relatively large number of embryos may need to be transfected for analysis. In addition, electroporation into the embryonic retina in ovo at later developmental stages (e.g., E4-E5) has experimental difficulties because the eyes are located in close proximity to the heart at those stages, which increases the risk of cardiac arrest after electroporation.
The system described here allows rapid and robust gene suppression in the developing chick retina. This approach can also be applied to other parts of the chick embryo, including the neural and non-neural tissues. We, therefore, expect that this system can contribute to the elucidation of molecular mechanisms of chick development.

The vertebrate retina is an important model system for studying neural development. Despite its peripheral location, the retina is anatomically and developmentally an extension of the central nervous system, and the optic nerve, which consists of axons of retinal ganglion cells, represents a tract within the central nervous system. The chick retina has significant advantages as a model system to study the molecular mechanism of neural development: It is large and develops rapidly; it has structural and functional similarities to the human retina; it is highly accessible for visualization and experimental manipulations. Molecular mechanisms of cell proliferation and differentiation, morphogenesis, and axon guidance during neural development have been extensively studied by using the chicken retina.
In ovo electroporation has been successfully used over the last two decades to introduce ectopic genes into cells in the developing chick embryo. This technique allows for labeling of developing cells, cell fate tracing, and tracing of cell migration and axon tracts, as well as ectopic gene expression for in vivo analysis of gene function. The conditions of in ovo electroporation for efficient ectopic gene expression in chick embryos have been well established1,2,3.
Despite these advantages, the lack of a stable loss-of-function technique for studies of gene function had been a major technical limitation of the chick embryo. Whereas chick embryos electroporated with small interfering RNAs (siRNAs)4 or expression vectors for short hairpin RNAs (shRNAs)5 show knockdown of the targeted gene, gene suppression in those approaches is transient because the effects disappear once cells lose the introduced RNAs or DNAs. A more stable gene suppression can be achieved by delivering siRNAs into chick embryos by an RCAS (Replication Competent Avian sarcoma-leukosis virus (ASLV) long terminal repeat (LTR) with a Splice acceptor) retrovirus system6. The viral vector integrates into the host genome, and the ectopic genes are stably expressed. However, the retrovirus can only integrate into the genome of dividing cells during the mitotic (M) phase of the cell cycle, which may impose a limitation on the developmental stages and/or cell types for which this loss-of-function approach can be applied. In addition, expression of transgenes by RCAS appears slower and less robust than that induced by in ovo electroporation7.
Transposons are genetic elements that move from one location on the genome to another. The Tol2 element is a member of the hAT transposable element family and contains an internal gene encoding a transposase that catalyzes the transposon reaction of the Tol2 element8. When a plasmid vector that carries a gene expression cassette flanked by the sequences of the left and right ends of the Tol2 elements (200 bp and 150 bp, respectively) is introduced into vertebrate cells with a Tol2 transposase expression construct, the expression cassette is excised from the plasmid and integrated into the host genome, which supports a stable expression of the ectopic gene (Figure 1). It has been shown that the Tol2 transposable element can induce gene transposition very efficiently in different vertebrate species, including zebrafish9,10, frogs11, chicks12, and mice13, and thus is a useful method of transgenesis and insertional mutagenesis. The Tol2 transposon system has been successfully used for conditional knockdown of a target gene by genomic integration of siRNA that is processed from long double-stranded RNA14.
This protocol describes a loss-of-function approach in the chick embryo that involves the introduction of artificial microRNAs (miRNAs) by the Tol2 transposon system15,16. In this approach, an expression cassette for the EmGFP (emerald green fluorescent protein) marker and artificial miRNAs against a target gene is cloned into a Tol2 transposon vector. The Tol2 transposon construct is then 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 studies, we successfully knocked down the expression of Nel, an extracellular glycoprotein predominantly expressed in the nervous system, in the developing chick retina (see Representative Results). Our results indicate that stable and efficient gene suppression can be achieved in ovo by this technique.

<table><tbody><tr><td>18 G needle, 2&quot;</td><td>VWR</td><td>89219-320</td><td></td></tr><tr><td>AP-TAG kit A and AP-TAG kit B</td><td>GenHunter Corp</td><td>Q201 and Q202</td><td>Plasmid vectors for making AP fusion proteins (https://www.genhunter.com/products/ap-tag-kit-a.html, https://www.genhunter.com/products/ap-tag-kit-b.html)</td></tr><tr><td>Block-iT RNAi Designer</td><td>Invitrogen</td><td></td><td>An online tool to choose target sequences and design pre-miRNA sequences (https://rnaidesigner.thermofisher.com/rnaiexpress/)</td></tr><tr><td>BSA 10 mg</td><td>Sigma-Aldrich</td><td>A2153</td><td></td></tr><tr><td>C115CB cables</td><td>Sonidel</td><td>C115CB</td><td>https://www.sonidel.com/product_info.php?products_id&amp;frac14;254</td></tr><tr><td>C117 cables</td><td>Sonidel</td><td>C117</td><td>https://www.sonidel.com/product_info.php?products_id&amp;frac14;252</td></tr><tr><td>Capillary tubes with omega dot fiber (Micropipette needles)</td><td>FHC</td><td>30-30-1</td><td>1 mm O.D. 0.75 mm I.D</td></tr><tr><td>CUY21 square wave electroporator</td><td>Nepa Gene</td><td>CUY21</td><td></td></tr><tr><td>Diethanolamine (pH 9.8)</td><td>Sigma-Aldrich</td><td>D8885</td><td></td></tr><tr><td>Dissecting microscope</td><td></td><td></td><td></td></tr><tr><td>Egg incubator</td><td>Kurl</td><td>B-Lab-600-110</td><td>https://www.flemingoutdoors.com/kuhl%2D%2D-600-egglaboratory-incubator%2D%2D-b-lab-600-110.html</td></tr><tr><td>Electrode holder</td><td>Sonidel</td><td>CUY580</td><td>https://www.sonidel.com/product_info.php?products_id&amp;frac14;85</td></tr><tr><td>Electrodes</td><td>Nepa Gene</td><td>CUY611P3-1</td><td>https://www.sonidel.com/product_info.php?products_id&amp;frac14;94</td></tr><tr><td>Electromax DH10B</td><td>Invitrogen</td><td>18290-015</td><td>Electrocompetent E. coli cells</td></tr><tr><td>Fast green FCF</td><td>Sigma-Aldrich</td><td>F7258</td><td></td></tr><tr><td>Fertilized chicken eggs (Gallus gallus)</td><td>Obtained from commercial vendors (e.g. Charles River) or local farmers</td><td></td><td></td></tr><tr><td>Gooseneck fiber light source</td><td></td><td></td><td></td></tr><tr><td>FuGene 6 transfection reagent</td><td>Promega</td><td>E2691</td><td></td></tr><tr><td>Hamilton syringe (50 &amp;mu;L)</td><td>Sigma-Aldrich</td><td>20715</td><td>Hamilton Cat No&amp;nbsp; 80901</td></tr><tr><td>Hanks&#039; balanced salt solution</td><td>Sigma-Aldrich</td><td>H6648</td><td></td></tr><tr><td>Heavy mineral oil</td><td>Sigma-Aldrich</td><td>330760</td><td></td></tr><tr><td>HEPES</td><td>GIBCO</td><td>15630080</td><td></td></tr><tr><td>L-Homoarginine</td><td>Sigma-Aldrich</td><td>H10007</td><td></td></tr><tr><td>MgCl2</td><td>Sigma-Aldrich</td><td>13112</td><td></td></tr><tr><td>Micromanipulator</td><td>Narishige (Japan)</td><td>MM3</td><td>http://products.narishige-group.com/group1/MM-3/electro/english.html</td></tr><tr><td>Micropipette puller</td><td>Shutter Instrument</td><td>P97</td><td></td></tr><tr><td>p-Nitrophenylphosphate</td><td>Sigma-Aldrich</td><td>20-106</td><td></td></tr><tr><td>PBS</td><td>Sigma-Aldrich</td><td>D8662</td><td></td></tr><tr><td>pCAGGS-T2TP vector</td><td></td><td></td><td>Tol2 transposase expression plasmid. A generous kind gift of Koichi Kawakami (National Institute of Genetics, Japan). Also available from Addgene.</td></tr><tr><td>Pfu</td><td>ThermoFisher</td><td>F566S</td><td></td></tr><tr><td>Picospritzer (Optional)</td><td>Parker</td><td></td><td>Pressure microinjection system</td></tr><tr><td>Plasmid maxi kit</td><td>Qiagen</td><td>12163</td><td>Plasmid maxiprep kit</td></tr><tr><td>pT2K-CAGGS vector</td><td></td><td></td><td>Tol2 transposon vector. Kindly provided by Yoshiko Takahashi (Kyoto University, Japan)</td></tr><tr><td>PVC tubing</td><td>VWR (UK)</td><td>228-3830</td><td></td></tr><tr><td>Spectinomycin</td><td>Sigma-Aldrich</td><td>S9007-5</td><td></td></tr><tr><td>T4 DNA ligase</td><td>Promega</td><td>M1801</td><td></td></tr><tr><td>The BLOCK-iT Pol II miR RNA expression kit with EmGFP</td><td>Invitrogen</td><td>K493600</td><td>Contains the miRNA expression vector (pcDNA6.2-GW/EmGFP-miRNA), a control vector (pcDNA6.2-GW/EmGFP-miRNA-negative control plasmid), accessory reagents, and instructions (https://www.thermofisher.com/order/catalog/product/K493600?SID.srch-hj-K4936-00)</td></tr><tr><td>Thermal cycler</td><td></td><td></td><td></td></tr></tbody></table>

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.

Construction of Tol2 transposon constructs for expression of artificial miRNAs against Nel 
Nel (Neural Epidermal growth factor (EGF)-Like; also known as Nell2) is an extracellular glycoprotein. It has structural similarities with thrombospondin-1 and is predominantly&#160;expressed in the nervous system20,21. We have previously demonstrated that Nel regulates differentiation and survival of retinal ganglion cells15 and acts as an inhibitory guidance cue for retinal axons22,23,24. In the developing chick retina, Nel expression is detected in the presumptive pigment epithelium at HH15 (embryonic day (E) 2.5). At HH20 (E3.5), Nel expression is also observed in newly differentiated retinal ganglion cells. Strong expression of Nel in the pigment epithelium and retinal ganglion cells continues at least until E1815.
To examine the function of Nel in the development of retinal ganglion cells, expression of the Nel gene was knocked down by introducing artificial miRNAs into the developing retina using in ovo electroporation and the Tol2 transposon system15,16. First, pairs of single-stranded DNA oligonucleotides were designed for 10 potential target sequences in the protein-coding region of the chicken Nel cDNA (GenBank accession number: NM_001030740.1) by using the online RNAi designing tool (see Table of Materials). The oligonucleotide pairs were annealed and individually cloned into the miRNA expression vector. Then, individual constructs were transfected into HEK293T cells that stably express Nel-AP, and their knockdown efficiencies were evaluated by measuring AP activity in the culture media. The pcDNA6.2-GW/EmGFP-miRNA-negative control plasmid was used as a control (Figure 4A).
Three Nel pre-miRNA sequences that showed highest knock down effects were selected (against nucleotides 482-502, 910-930, and 2461-2481 of chicken Nel cDNA, respectively), and two pre-miRNA sequences were tandemly cloned into the miRNA expression vector (pcDNA6.2-GW/EmGFP-2x Nel pre-miRNA), according to the manufacturer&#39;s instructions17. The expression cassettes encoding the two pre-miRNA and EmGFP were amplified by PCR with an artificial EcoRI site on both ends (5&#8242;-GGGAATTCTCTGGCTAACTAGAGAAC-3&#8242; and 5&#8242;-CCGAATTCCCTCTAGATCAACCACT-3&#8242;) and cloned into the pT2K-CAGGS vector (pT2K-CAGGS-EmGFP-2x Nel pre-miRNA). The sequence of the expression cassette was confirmed by using the primers used for the PCR. pT2K-CAGGS-EmGFP-2x Nel pre-miRNA constructs were individually co-transfected with the Tol2 transposase expression vector (pCAGGS-T2TP) into HEK293T cells that stably express Nel-AP, and inhibitory effects on Nel-AP expression was evaluated by measuring the AP activities in the culture media. As shown in Figure 4B, knockdown efficiency was significantly enhanced by the chaining of two miRNA sequences. Robust suppression of Nel-AP expression (more than 90%) was observed at least until 13 days after transfection (Figure 4B).
Stable suppression of Nel expression in the developing chick retina 
A pT2K-CAGGS-EmGFP-2x Nel pre-miRNA construct (containing target sequences for nucleotides 482-502 and 2461-2481 of the chicken Nel cDNA) was co-transfected with the transposase expression vector (pCAGGS-T2TP) into the temporal or nasal side of the chick retina by in ovo electroporation at HH9-11 (Figure 3, Figure 5A). Sections were prepared from E4.5 or E8 retinae, and effects on Nel expression was evaluated by immunohistochemistry using anti-Nel antibody22. At E4.5, the expression of Nel in the retinal pigment epithelium was significantly reduced in EmGFP expressing cells (Figure 5B-D). At E8, decreases in Nel expression were also clearly observed in retinal ganglion cells (Figure 5E-I). Significant suppression of Nel expression continued at least until E18 (data not shown). The introduction of control miRNA did not affect Nel expression in the retina (Figure 5J-O).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62399/62399fig01.jpg" /> 
Figure 1: Transposition of Tol2-flanked cDNAs by transposase. A schematic showing transposition of a Tol2-flanked expression cassette for EmGFP and chained two pre-miRNA sequences (2x pre-miRNA) by transposase. When a Tol2 transposon vector containing the expression cassette (pT2K-CAGGS-EmGFP-2x miRNA construct) is introduced into cells with a Tol2 transposase expression construct (pCAGGS-T2TP), the Tol2-flanked expression cassette is excised from the vector and integrated into the host genome by the transposase activity. Expression of EmGFP and miRNAs is driven by the ubiquitous promoter CAGGS. This figure has been modified from Nakamoto et al.15. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62399/62399fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62399/62399fig2v2.jpg" /> 
Figure 2: Structure of the engineered pre-miRNA. (A) The designed pre-miRNA sequence is based on the murine miR-155 sequence17. The top strand oligo contains (from the 5&#39; end to the 3&#39; end) a four-nucleotide overhang (TGCT (blue)), antisense target sequence (21 nucleotides), terminal loop sequence (red), and sense target sequence (19 nucleotides). The antisense target sequence represents the mature miRNA sequence. The bottom strand oligo contains a four-nucleotide 5&#39; overhang (CCTG (blue)) and the reverse complement sequence of the top strand oligo but lacks the four-nucleotide overhang at the 3&#39; end (AGCA-3&#39;: reverse complement of 5&#39;-TGCT of the top-strand oligo). The sense target sequence lacks nucleotides 9 and 10 of the target sequence. The &#34;extra&#34; two nucleotides in the antisense target sequence form an internal loop in the stem-loop structure of the mature miRNA molecule, which results in a higher knockdown rate17. (B) An example of target sequence against chicken Nel. Sense and antisense sequences in the top and bottom strands for a target&#160;sequence of the chicken Nel gene (GenBank accession number NM_001030740.1, nucleotides 482-502) are shown. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62399/62399fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62399/62399fig03.jpg" /> 
Figure 3: In ovo microinjection and electroporation. (A) Workstation for in ovo microinjection and electroporation. (1) Dissecting microscope. (2) Injection apparatus. (3) Electroporation apparatus. (4) Dual gooseneck microscope illuminator. (5) Electroporator. (B) Injection apparatus. A Hamilton syringe (6) with an 18 G needle (7) is connected to PVC tubing (8) and to a pulled micropipette needle (9) and set on a micromanipulator (10). The inner space of the apparatus is filled with heavy mineral oil. (C) Electroporation apparatus. A set of platinum electrodes (11) with an electrode holder (12) are set on a micromanipulator (13). The electrodes are connected to the electroporator with cables (14). (D) Microinjection of DNA cocktail solution into the optic vesicle at HH 10-11. The pulled micropipette needle is inserted into the optic vesicle from its proximal side.DNA cocktail solution with fast green&#160;(blue) is injected into the optic vesicle until the dye fills the entire lumen. (E) Electroporation into the optic vesicle. Electrodes are placed in parallel (The distance between the electrodes is 2 mm) and in a way so that the injected optic vesicle is located between the electrodes: One electrode is located near the anterior (nasal) surface of the optic vesicle, and the other is in the posterior part of the embryo at the level of the hindbrain. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62399/62399fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62399/62399fig04.jpg" /> 
Figure 4: In vitro evaluation of individual or chained pre-miRNA sequences for Nel gene suppression efficiency. (A) Knockdown effects of individual pre-miRNA sequences. Results of five representative constructs (Antisense target sequences corresponding nucleotide numbers 482-502, 910-930, 1106-1126, 1663-1683, and 2461-2481 of chicken Nel cDNA (GenBank accession number: NM_001030740.1)) are shown. Individual miRNA expression constructs (pcDNA6.2-GW/EmGFP-Nel pre-miRNA) were transfected into HEK293T cells that stably express Nel-AP. The pcDNA6.2-GW/EmGFP-miRNA-negative control plasmid was used as a control. Expression of Nel-AP was evaluated by measuring the AP activities in the culture media 48-96 h after transfection. Three Nel pre-miRNA expression constructs (against nucleotides 482-502, 910-930, and 2461-2481, respectively) showed significant suppression of Nel expression. (B) Knockdown effects of chained pre-miRNA sequences. Two of the three most potent pre-miRNA sequences (against nucleotides 482-502, 910-930, and 2461-2481) were tandemly cloned into the pcDNA6.2-GW/EmGFP-miR vector, and the expression cassette (containing the two pre-miRNA sequences and EmGFP cDNA) was transferred to the pT2K-CAGGS vector (pT2K-CAGGS-EmGFP-2x Nel pre-miRNA). The pT2K-CAGGS-EmGFP-2x Nel pre-miRNA constructs were individually co-transfected with expression vectors for Tol2 transposase (pCAGGS-T2TP) into HEK293T cells that stably express Nel-AP, and expression of Nel-AP was evaluated by measuring the AP activities in the culture media from 2 to 13 days after transfection. All three combinations (482-502/910-930, 482-502/2461-2481, 910-930/2461-2481) showed enhanced suppression activities compared to unchained individual pre-miRNA sequences. Significant suppression of Nel-AP expression was observed from day 2 to at least day 13. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62399/62399fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62399/62399fig05.jpg" /> 
Figure 5: Stable knockdown of Nel expression in the developing chick retina by Tol2 transposon-mediated integration of miRNA transgene. A Tol2 transposon construct containing an expression cassette of two Nel pre-miRNA sequences and EmGFP (pT2K-CAGGS-EmGFP-Nel pre-miRNA (482-502)-Nel pre-miRNA (2461-2481)) (A-I) or a control construct that expresses an unrelated miRNA and EmGFP (J-O) was co-transfected with a transposase expression vector (pCAGGS-T2TP) into the temporal or nasal half of the optic vesicle by in ovo electroporation at HH9-HH11 (E1.5). Retinal sections were prepared at E4.5 (B-D, J-L) or E8 (E-I, M-O), and efficiencies of Nel gene suppression were examined by immunohistochemistry using anti-Nel antibody (red). Since DNA is negatively charged, the transgenes are electroporated into the tissue on the anode side; thus, only half of the retina was labeled with EmGFP (transfection (TF) side, green). (A) Expression of the EmGFP marker gene in the temporal half of the retina at E4.5. The optic fissure (white arrow) represents the boundary between transfected and untransfected (control) sides. (B-I) RNAi knockdown of Nel expression in the developing chick retina. (B-D) At E4.5, Nel expression is significantly reduced (B,D) in retinal pigment epithelium (PE) cells that express EmGFP (C,D). Note the complementary pattern of Nel immunostaining and EmGFP expression in D. NR, neural retina. (E-I) At E8, Nel expression (E,G) in the retinal pigment epithelium and the ganglion cell layer (GCL) is decreased on the transfection side (F,G). (H, I) Higher magnification views of a transfection area and a corresponding control area in E (indicated by small rectangles), respectively. (D,G) Merged images of the left two images. (J-O) Expression of control miRNA. Introduction of the negative control construct does not affect the expression of Nel in E4.5 (J-L) or E8 (M-O) retina. (L,O) Merged images of the left two images. The boundary between transfection and control sides is indicated by a dotted line in C, F, K, and N. Scale bars, 50 &#181;m. (A) and (B-I) have been modified from Nakamoto, M. &#38; Nakamoto, C16 and Nakamoto et al.15, respectively. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62399/62399fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">Annealing reaction setup</td>
		</tr>
		<tr>
			<td>Top strand oligo (200 &#956;M in TE buffer)</td>
			<td>5 &#956;L (Final concentration 50 &#956;M)</td>
		</tr>
		<tr>
			<td>Bottom strand oligo (200 &#956;M in TE buffer)</td>
			<td>5 &#956;L (Final concentration 50 &#956;M)</td>
		</tr>
		<tr>
			<td>10x Oligo annealing buffer*</td>
			<td>2 &#956;L</td>
		</tr>
		<tr>
			<td>DNase/RNase-free water*</td>
			<td>8 &#956;L</td>
		</tr>
		<tr>
			<td>Total</td>
			<td>20 &#956;L</td>
		</tr>
		<tr>
			<td colspan="2">* Provided in the miRNA expression kit (see Table of Materials)</td>
		</tr>
	</tbody>
</table>
Table 1: Annealing reaction setup 
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">2x AP substrate buffer</td>
		</tr>
		<tr>
			<td>10 M Diethanolamine (pH 9.8)</td>
			<td>4 mL</td>
		</tr>
		<tr>
			<td>1 M MgCl2</td>
			<td>10 &#956;L</td>
		</tr>
		<tr>
			<td>L-Homoarginine</td>
			<td>45 mg</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>10 mg</td>
		</tr>
		<tr>
			<td>p-Nitrophenylphosphate</td>
			<td>63 mg</td>
		</tr>
		<tr>
			<td>H2O</td>
			<td>Add to make up a total volume of 20 mL</td>
		</tr>
	</tbody>
</table>
Table 2: 2x AP substrate buffer. The solution should be stored in aliquots at -20 &#176;C and be protected from light as p-nitrophenylphosphate is light sensitive.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="4">Reaction setup</td>
		</tr>
		<tr>
			<td colspan="3">pcDNA6.2-GW/EmGFP-2x pre-miRNA plasmid (From Section 1.5) 
			or pcDNA6.2-GW/EmGFP-miRNA-negative control plasmid* (0.1 &#956;g/&#956;L)</td>
			<td>1 &#956;L</td>
		</tr>
		<tr>
			<td colspan="3">10x Pfu buffer</td>
			<td>5 &#956;L</td>
		</tr>
		<tr>
			<td colspan="3">dNTP mix (10 mM each of dATP, dCTP, dGTP, and dTTP)</td>
			<td>1 &#956;L</td>
		</tr>
		<tr>
			<td colspan="3">EmGFP forward primer* (5&#8217;-GGCATGGACGAGCTGTACAA-3&#8217;; 10 &#956;M)</td>
			<td>1 &#956;L</td>
		</tr>
		<tr>
			<td colspan="3">miRNA reverse primer* (5&#8217;- CTCTAGATCAACCACTTTGT-3&#8217;; 10 &#956;M)</td>
			<td>1 &#956;L</td>
		</tr>
		<tr>
			<td colspan="3">Pfu DNA polymerase (5 units/&#956;L)</td>
			<td>0.5 &#956;L</td>
		</tr>
		<tr>
			<td colspan="3">H2O</td>
			<td>40.5 &#956;L</td>
		</tr>
		<tr>
			<td colspan="3">Total volume</td>
			<td>50&#160; &#956;L</td>
		</tr>
		<tr>
			<td colspan="3">* Provided in the miRNA expression kit.</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Thermocycling conditions</td>
		</tr>
		<tr>
			<td colspan="3">94 &#176;C</td>
			<td>5 min</td>
		</tr>
		<tr>
			<td colspan="3">30 cycles of the following:</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="3">&#160;&#160;&#160;&#160; 94 &#176;C</td>
			<td>45 s</td>
		</tr>
		<tr>
			<td colspan="3">&#160;&#160;&#160;&#160; 58 &#176;C</td>
			<td>1 min</td>
		</tr>
		<tr>
			<td colspan="3">&#160;&#160;&#160;&#160; 72 &#176;C</td>
			<td>2 min</td>
		</tr>
		<tr>
			<td colspan="3">72 &#176;C</td>
			<td>10 min</td>
		</tr>
	</tbody>
</table>
Table 3: Reaction setup and the thermocycling conditions.

Watch this Scientific Journal Video about Loss-of-Function Approach in the Embryonic Chick Retina by Using Tol2 Transposon-Mediated Transgenic Expression of Artificial microRNAs at JoVE.com

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

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 ...

loss-function-approach-embryonic-chick-retina-using-tol2-transposon

Nanyang Technological University

Research

JoVE Journal

Neuroscience

983 Views.  Valparaiso University. Deze methodologie biedt een verlies-van-functiebenadering voor studies van retinale ontwikkeling bij het kuiken. Deze techniek ondersteunt snelle en aanhoudende onderdrukking van doelwitgenen in een specifiek gebied van het netvlies. Deze aanpak kan ook worden toegepast op andere delen van het kuikenembryo, waaronder zowel de neurale als niet-neurale weefsels.Bereid voor in ovo-elektroporatie een 0,25% Fast Green-oplossing door 25 milligram Fast Green FCF op te lossen in 10 milliliter ...

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

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 ...

Developmental Biology

Loss- and Gain-of-function Approach to Investigate Early Cell Fate Determinants in Preimplantation Mouse Embryos

Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene modification in preimplantation embryos provides a means to study the underlying mechanisms of genes implicated in, for instance, cellular differentiation into the trophectoderm (TE) and the inner cell mass (ICM). Here, we describe a protocol that examines the role of neogenin as an authentic receptor for initial cell fate determination in preimplantation mouse embryos. First, we discuss the experimental manipulations that were used to produce gain and loss of neogenin function by microinjecting neogenin cDNA and shRNA; the effectiveness of this approach was confirmed by a strong correlation between the pair-wise expression levels of either red fluorescent protein (RFP) or green fluorescent protein (GFP) and the immunocytochemical quantification of neogenin expression. Secondly, overexpression of neogenin in preimplantation mouse embryos leads to normal ICM development while neogenin knockdown causes the ICM to develop abnormally, implying that neogenin could be a receptor that relays extracellular cues to drive blastomeres to early cell fates. Given the success of this detailed protocol in investigating the function of a novel embryonic developmental stage-specific receptor, we propose that it has the potential to aid in exploration and identification of other stage-specific genes during embryogenesis.

The goal of this protocol is to describe a loss- and gain-of function method that is applicable to identify neogenin as a stage-specific receptor that leads to trophectoderm and inner cell mass differentiation in preimplantation mouse embryos.

 Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene ...

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 ...

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced in vivo throughout the rapid developmental period. Multiple processes can be studied including cell proliferation, gene expression, cell migration and morphogenesis. Importantly, the generation of chimeras through transplantations can be easily performed, allowing mosaic labeling and tracking of individual cells under the influence of the host environment. For example, by combining functional gene manipulations of the host embryo (e.g., through morpholino microinjection) and live imaging, the effects of extrinsic, cell nonautonomous signals (provided by the genetically modified environment) on individual transplanted donor cells can be assessed. Here we demonstrate how this approach is used to compare the onset of fluorescent transgene expression as a proxy for the timing of cell fate determination in different genetic host environments.
In this article, we provide the protocol for microinjecting zebrafish embryos to mark donor cells and to cause gene knockdown in host embryos, a description of the transplantation technique used to generate chimeric embryos, and the protocol for preparing and running in vivo time-lapse confocal imaging of multiple embryos. In particular, performing multiposition imaging is crucial when comparing timing of events such as the onset of gene expression. This requires data collection from multiple control and experimental embryos processed simultaneously. Such an approach can easily be extended for studies of extrinsic influences in any organ or tissue of choice accessible to live imaging, provided that transplantations can be targeted easily according to established embryonic fate maps.

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

Live tracking of individual WT retinal progenitors in distinct genetic backgrounds allows for the assessment of the contribution of cell non-autonomous signaling during neurogenesis. Here, a combination of gene knockdown, chimera generation via embryo transplantation and in vivo time-lapse confocal imaging was utilized for this purpose.

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced ...

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 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.

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.

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

In ovo Expression of MicroRNA in Ventral Chick Midbrain

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial and temporal control of ectopic microRNA expression. However, ventral brain structures like ventral midbrain are rather difficult to reach for any manipulations. Here, we demonstrate an efficient way to electroporate miRNA into ventral midbrain using thin platinum electrodes. This method offers a reliable way to transfect specific areas of the midbrain and a useful tool for in vivo studies.

In ovo Expression of MicroRNA in Ventral Chick Midbrain

Ectopic expression is one technique to elucidate the microRNAs role in brain development. However, targeting specific areas using in ovo electroporation is challenging. Here, we show an efficient way to selectively electroporate ventral and dorsal midbrain regions.

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

Biology

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 ...

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a workflow on how to combine artificial microRNA (miR)-mediated RNA interference with optogenetics to achieve selective stimulation of manipulated presynaptic boutons in acute brain slices. The experimental approach involves the use of a single viral construct and a single neuron-specific promoter to drive the expression of both an optogenetic probe and artificial miR(s) against presynaptic gene(s). When stereotactically injected in the brain region of interest, the expressed construct makes it possible to stimulate with light exclusively the neurons with reduced expression of the gene(s) under investigation. This strategy does not require the development and maintenance of genetically modified mouse lines and can in principle be applied to other organisms and to any neuronal gene of choice. We have recently applied it to investigate how the knockdown of alternative splice isoforms of presynaptic P/Q-type voltage-gated calcium channels (VGCCs) regulates short-term synaptic plasticity at CA3 to CA1 excitatory synapses in acute hippocampal slices. A similar approach could also be used to manipulate and probe the neuronal circuitry in vivo.

This protocol provides a workflow on how to combine artificial microRNA-mediated RNA interference with optogenetics to stimulate specifically presynaptic boutons with reduced expression of selective gene(s) within intact neuronal circuits.

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a ...

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

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