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...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>18 G needle, 2&#34;</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&#188;254</td>
		</tr>
		<tr>
			<td>C117 cables</td>
			<td>Sonidel</td>
			<td>C117</td>
			<td>https://www.sonidel.com/product_info.php?products_id&#188;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&#188;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&#188;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 &#956;L)</td>
			<td>Sigma-Aldrich</td>
			<td>20715</td>
			<td>Hamilton Cat No&#160; 80901</td>
		</tr>
		<tr>
			<td>Hanks&#39; 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>
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		<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 cells<sup cl...

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

Research

JoVE Journal

Neuroscience

969 Views.  Valparaiso University. 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.

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

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to cis-regulatory elements (CREs), short (&tilde;300-600 bp) stretches of genomic DNA which can lie upstream, downstream, or within the introns of the genes they control. CREs (i.e., enhancers/promoters) typically consist of multiple clustered binding sites for both transcriptional activators and repressors1-3. They serve as logical integrators of transcriptional input giving a unitary output in the form of spatiotemporally precise and quantitatively exact promoter activity. Most studies of mammalian cis-regulation to date have relied on mouse transgenesis as a means of assaying the enhancer function of CREs4-5. This technique is time-consuming, costly and, on account of insertion site effects, largely non-quantitative. On the other hand, quantitative assays for mammalian CRE function have been developed in tissue culture systems (e.g., dual luciferase assays), but the in vivo relevance of these results is often uncertain. 
Electroporation offers an excellent alternative to traditional mouse transgenesis in that it permits both spatiotemporal and quantitative assessment of cis-regulatory activity in living mammalian tissue. This technique has been particularly useful in the analysis of cis-regulation in the central nervous system, especially in the cerebral cortex and the retina6-8. While mouse retinal electroporation, both in vivo and ex vivo, has been developed and extensively described by Matsuda and Cepko6-7,9, we have recently developed a simple approach to quantify the activity of photoreceptor-specific CREs in electroporated mouse retinas10. Given that the amount of DNA that is introduced into the retina by electroporation can vary from experiment to experiment, it is necessary to include a co-electroporated 'loading control' in all experiments. In this respect, the technique is very similar to the dual luciferase assay used to quantify promoter activity in cultured cells. 
When assaying photoreceptor cis-regulatory activity, electroporation is usually performed in newborn mice (postnatal day 0, P0) which is the time of peak rod production11-12. Once retinal cell types become post-mitotic, electroporation is much less efficient. Given the high rate of rod birth in newborn mice and the fact that rods constitute more than 70% of the cells in the adult mouse retina, the majority of cells that are electroporated at P0 are rods. For this reason, rod photoreceptors are the easiest retinal cell type to study via electroporation. The technique we describe here is primarily useful for quantifying the activity of photoreceptor CREs.

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

This protocol describes a simple and inexpensive way to quantify the activity of cis-regulatory elements (i.e., enhancer/promoters) in living mouse retinas via explant electroporation. DNA preparation, retinal dissection, electroporation, retinal explant culture, and post-fixation analysis and quantification are described.

Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to ...

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 ...

Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

Electroporation of the chick embryonic neural tube has many advantages such as being quick and efficient for the expression of foreign genes into neuronal cells. In this manuscript we provide a method that demonstrates uniquely how to electroporate DNA into the avian hindbrain at E2.75 in order to specifically label a subset of neuronal progenitors, and how to follow their axonal projections and synaptic targets at much advanced stages of development, up to E14.5. We have utilized novel genetic tools including specific enhancer elements, Cre/Lox - based plasmids and the PiggyBac-mediated DNA transposition system to drive GFP expression in a subtype of hindbrain cells (the dorsal most subgroup of interneurons, dA1). Axonal trajectories and targets of dA1 axons are followed at early and late embryonic stages at various brainstem regions. This strategy contributes advanced techniques for targeting cells of interest in the embryonic hindbrain and for tracing circuit formation at multiple stages of development.

How neuronal networks are established in the embryonic brain is a fundamental question in developmental neurobiology. Here we combined an electroporation technique with novel genetic tools, such as Cre/Lox&#x2013;plasmids and PiggyBac-mediated DNA transposition system in the avian hindbrain to label dorsal interneurons and track their axonal projections and synaptic targets at various developmental stages.

Electroporation of  the chick embryonic neural tube has many advantages such as being quick and efficient  for the expression of foreign genes into ...

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the use of IUE for investigating behavior or neuropathology in the adult brain has been limited by insufficient methods for monitoring of IUE transfection success by non-invasive techniques in postnatal animals. 
 For the present study, E16 rats were used for IUE. After intraventricular injection of the nucleic acids into the embryos, positioning of the tweezer electrodes was critical for targeting either the developing cortex or the hippocampus. 
 Ventricular co-injection and electroporation of a luciferase gene allowed monitoring of the transfected cells postnatally after intraperitoneal luciferin injection in the anesthetized live P7 pup by in vivo bioluminescence, using an IVIS Spectrum device with 3D quantification software. 
 Area definition by bioluminescence could clearly differentiate between cortical and hippocampal electroporations and detect a signal longitudinally over time up to 5 weeks after birth. This imaging technique allowed us to select pups with a sufficient number of transfected cells assumed necessary for triggering biological effects and, subsequently, to perform behavioral investigations at 3 month of age. As an example, this study demonstrates that IUE with the human full length DISC1 gene into the rat cortex led to amphetamine hypersensitivity. Co-transfected GFP could be detected in neurons by post mortem fluorescence microscopy in cryosections indicating gene expression present at &ge;6 months after birth.
 We conclude that postnatal bioluminescence imaging allows evaluating the success of transient transfections with IUE in rats. Investigations on the influence of topical gene manipulations during neurodevelopment on the adult brain and its connectivity are greatly facilitated. For many scientific questions, this technique can supplement or even replace the use of transgenic rats and provide a novel technology for behavioral neuroscience.

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

Genes can be manipulated during development of the cortex or the hippocampus of the rat via in utero electroporation (IUE) at E16, to enable fast and targeted modifications in neuronal connectivity for later studies of behavior or neuropathology in adult animals. Postnatal in vivo imaging for control of IUE success is performed by bioluminescence of activating co-transfected luciferase.

 In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the ...

Micromanipulation of Gene Expression in the Adult Zebrafish Brain Using Cerebroventricular Microinjection of Morpholino Oligonucleotides

Manipulation of gene expression in tissues is required to perform functional studies. In this paper, we demonstrate the cerebroventricular microinjection (CVMI) technique as a means to modulate gene expression in the adult zebrafish brain. By using CVMI, substances can be administered into the cerebroventricular fluid and be thoroughly distributed along the rostrocaudal axis of the brain. We particularly focus on the use of antisense morpholino oligonucleotides, which are potent tools for knocking down gene expression in vivo. In our method, when applied, morpholino molecules are taken up by the cells lining the ventricular surface. These cells include the radial glial cells, which act as neurogenic progenitors. Therefore, knocking down gene expression in the radial glial cells is of utmost importance to analyze the widespread neurogenesis response in zebrafish, and also would provide insight into how vertebrates could sustain adult neurogenesis response. Such an understanding would also help the efforts for clinical applications in human neurodegenerative disorders and central nervous system regeneration. Thus, we present the cerebroventricular microinjection method as a quick and efficient way to alter gene expression and neurogenesis response in the adult zebrafish forebrain. We also provide troubleshooting tips and other useful information on how to carry out the CVMI procedure.

In this article, we demonstrate a method for manipulation of gene expression in the ventricular cells of the adult zebrafish telencephalon using antisense morpholino oligonucleotides. We present this method as an efficient and quick protocol that can be used for functional studies in the adult vertebrate brain.

Manipulation of gene expression in tissues is  required to perform functional studies. In this paper, we demonstrate the  cerebroventricular ...

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). It is increasingly being recognized that several neurodegenerative diseases such as Alzheimer&#8217;s, multiple sclerosis, and amyotrophic lateral sclerosis present elements of vascular compromise. In addition, the most prominent causes of blindness in pediatric and working age populations (retinopathy of prematurity and diabetic retinopathy, respectively) are characterized by vascular degeneration and failure of physiological vascular regrowth. The aim of this technical paper is to provide a detailed protocol to study CNS vascular regeneration in the retina. The method can be employed to elucidate molecular mechanisms that lead to failure of vascular growth after ischemic injury. In addition, potential therapeutic modalities to accelerate and restore healthy vascular plexuses can be explored. Findings obtained using the described approach may provide therapeutic avenues for ischemic retinopathies such as that of diabetes or prematurity and possibly benefit other vascular disorders of the CNS.

The rodent retina has long been recognized as an accessible window to the brain. In this technical paper we provide a protocol that employs the mouse model of oxygen-induced retinopathy to study the mechanisms that lead to failure of vascular regeneration within the central nervous system after ischemic injury. The described system can also be harnessed to explore strategies to promote regrowth of functional blood vessels within the retina and CNS.

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). ...

RNA-Seq Analysis of Differential Gene Expression in Electroporated Chick Embryonic Spinal Cord

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome wide analysis of differentially expressed transcripts after such an experimental manipulation has the potential to uncover an almost complete picture of the downstream effects caused by the transfected construct. This work describes a simple method for comparing transcriptomes from samples of transfected embryonic spinal cords comprising all steps between electroporation and identification of differentially expressed transcripts. The first stage consists of guidelines for electroporation and instructions for dissection of transfected spinal cord halves from HH23 embryos in ribonuclease-free environment and extraction of high-quality RNA samples suitable for transcriptome sequencing. The next stage is that of bioinformatic analysis with general guidelines for filtering and comparison of RNA-Seq datasets in the Galaxy public server, which eliminates the need of a local computational structure for small to medium scale experiments. The representative results show that the dissection methods generate high quality RNA samples and that the transcriptomes obtained from two control samples are essentially the same, an important requirement for detection of differential expression genes in experimental samples. Furthermore, one example is provided where experimental overexpression of a DNA construct can be visually verified after comparison with control samples. The application of this method may be a powerful tool to facilitate new discoveries on the function of neural factors involved in spinal cord early development.

This video shows the basic steps for performing whole transcriptome analysis on dissected chick embryonic spinal cord samples after transfection with in ovo electroporation.

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome ...

Using the Electroretinogram to Assess Function in the Rodent Retina and the Protective Effects of Remote Limb Ischemic Preconditioning

The ERG is the sum of all retinal activity. The ERG is usually recorded from the cornea, which acts as an antenna that collects and sums signals from the retina. The ERG is a sensitive measure of changes in retinal function that are pan-retinal, but is less effective for detecting damage confined to a small area of retina. In the present work we describe how to record the &#8216;flash&#8217; ERG, which is the potential generated when the retina is exposed to a brief light flash. We describe methods of anaesthesia, mydriasis and corneal management during recording; how to keep the retina dark adapted; electrode materials and placement; the range and calibration of stimulus energy; recording parameters and the extraction of data. We also describe a method of inducing ischemia in one limb, and how to use the ERG to assess the effects of this remote-from-the-retina ischemia on retinal function after light damage. A two-flash protocol is described which allows isolation of the cone-driven component of the dark-adapted ERG, and thereby the separation of the rod and cone components. Because it can be recorded with techniques that are minimally invasive, the ERG has been widely used in studies of the physiology, pharmacology and toxicology of the retina. We describe one example of this usefulness, in which the ERG is used to assess the function of the light-damaged retina, with and without a neuroprotective intervention; preconditioning by remote ischemia.

The electroretinogram (ERG) is an electrical potential generated by the retina in response to light. This paper describes how to use the ERG to assess retinal function, in dark-adapted rats, and how it can be can be used to assess a neuroprotective intervention, in the present case remote ischemic preconditioning.

The ERG is the sum of all retinal activity. The ERG is usually recorded from the cornea, which acts as an antenna that collects and sums signals from the ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

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

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Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

In ovo Expression of MicroRNA in Ventral Chick Midbrain

Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

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RNA-Seq Analysis of Differential Gene Expression in Electroporated Chick Embryonic Spinal Cord

Using the Electroretinogram to Assess Function in the Rodent Retina and the Protective Effects of Remote Limb Ischemic Preconditioning

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice