Name: In ovo Expression of MicroRNA in Ventral Chick Midbrain (Video) | JoVE Journal
Uploaded: 2013-09-16T07:00:00.000+00:00
Duration: 9 min 19 s
Description: Learn in ovo electroporation for targeted microRNA expression in chick ventral midbrain. This method enables efficient DNA delivery to mural progenitor cells for studying brain development.

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We acknowledge K. Mikic, who contributed to the initial phase of this movie and M. Nicolescu for the miR picture. C. Huber was supported by a fellowship of the IZKF of the Universtit&auml;tsklinikum T&uuml;bingen, A. Alwin Prem Anand by the Fort&uuml;ne programme of the Universtit&auml;tsklinikum T&uuml;bingen.

1. Requirements for In ovo Electroporation
<ol>
 <li>Preparing the vector construct: 
 miR sense and antisense primers were designed after the instructions of Ambion, annealed and ligated into ApaI/EcoRI digested pSilencer U6.1 vector. After successful transformation one of the resulting clones was grown and pSilencer plasmid was isolated by alkaline lysis method using a Quiagen midi preparation kit. </li>
 <li>Electrodes: 
 Electrodes can be purchased or easily be built13. We use self-made electrodes of platinum wire (Φ = 0.2, 0.25, or 0.5 mm in diameter) or in some cases an even smaller tungsten wire electrode (Φ = 0.1 mm)8 depending on which area we want to electroporate. To avoid scorching of tissue, we use as a rule the nearer to the tissue the thinner the electrodes. For broad electroporation of midbrain, we use 0.5 mm platinum wires soldered to a brass/stainless steel tubular shaft and fixed to an electrode holder with a distance of 3-5 mm (Figure 1). To electroporate specific areas, we use 0.25 mm platinum wire anodes and 0.2 mm platinum wire or 0.1 mm tungsten for the cathode with an approximate distance of 1 mm or less. </li>
 <li>Solutions: Have the following solutions ready: 
 <ol>
 <li>Autoclaved chicken Ringer, pH 7.5 (9.0 g NaCl, 0.42 g KCl, 0.24 g CaCl2, dissolve in 1 L distilled water).</li>
 <li>PBS (8 g NaCl, 0.2 g KCl, 0.24 g KH2PO4, 1.44 g Na2HPO4, dissolve in 1l distilled H2O). </li>
 <li>Fast Green: stock solution 10 mg/ml distilled H2O</li>
 <li> Slow drying ink (less shellac and thus less poisoning for the embryo). Note: A blue dichroic filter attached to a fiber optic lamp as described in Canto-Soler and Adler14 enhances the contrast and can eliminate the need to inject ink. </li>
 </ol>
 </li>
</ol>
<img src="/files/ftp_upload/50024/50024fig1.jpg" fo:content-width="6in" fo:src="/files/ftp_upload/50024/50024fig1highres.jpg" alt="Fiber optic sensor assembly diagram; experimental setup for measurements, optical fibers detail." data-altupdatedat="1787630408000"> 
Figure 1. Schematic diagram of electrodes and electrode holder. (A, B, C) Platinum wire was soldered to one side of a stainless steel tubular shaft. A hole was drilled into the other side of the tubular shaft thus that pin plugs can fit in. (A) Red and blue colored wires were connected to the pin plugs. At the opposite end the wire was fitted to banana connectors that work with the electrical sockets of the electroporator. (A, C) The platinum wire was bent in an angel of 90°. A layer of nail varnish isolated the shaft above the bend.
2. Preparations Directly Before In ovo Electroporation
<ol>
 <li>Prepare the following material:
 <ol>
 <li>Dilute Ink with chicken Ringer 1:6, add Antibiotic-Antimycotic solution (100x from Gibco) 1:100.</li>
 <li>Prepare vector solution: 1-2 μg/μl per specific DNA in H2O supplemented with Fast Green (1:20).</li>
 <li>Sterilize forceps, scissors, tungsten needle and electrodes with 70% ethanol.</li>
 <li>Pull micropipettes for nucleic acid injection from borosilicate glass capillaries (length to outer diameter: 100 mm x 0.9 mm). We use a simple puller PUL-1 (WPI, Berlin) with the settings shown in Table 1. </li>
 <li>Set the parameters of the electroporator as shown in Table 2.</li>
 </ol>
 </li> 
</ol>
 <table border="1">
 <tbody>
 <tr>
 <td fo:width="1in">Parameters</td>
 <td fo:width="1in">Values</td>
 </tr>
 <tr>
 <td>Heat </td>
 <td>350</td>
 </tr>
 <tr>
 <td>Pull</td>
 <td>100</td>
 </tr>
 <tr>
 <td>Velocity</td>
 <td>50</td>
 </tr>
 <tr>
 <td>Delay</td>
 <td>200</td>
 </tr>
 </tbody>
 </table>
Table 1. Settings for PUL-1 
 <table border="1">
 <tbody>
 <tr>
 <td fo:width="1.5in">Parameters</td>
 <td fo:width="1.5in">Values</td>
 </tr>
 <tr>
 <td>Frequency</td>
 <td>50 Hz</td>
 </tr>
 <tr>
 <td>Delay</td>
 <td>20 - 50 msec*</td>
 </tr>
 <tr>
 <td>Width</td>
 <td>20 msec</td>
 </tr>
 <tr>
 <td>Voltage</td>
 <td>8-25 V *</td>
 </tr>
 <tr>
 <td>Pulse</td>
 <td>3 - 5 times</td>
 </tr>
 <tr>
 <td colspan="2">*Depending on electrode diameter and distance. Please take that into account when changing one of the parameters. </td>
 </tr>
 </tbody>
 </table>
Table 2. Settings for the Pulse Stimulator
<ol start="2">
 <li>Prepare chick embryos for electroporation:
 <ol>
 <li>Incubate fertilized chicken eggs on their longer side at 37 °C with 65% humidity for 39 hr to acquire embryos between Hamburger &amp; Hamilton (HH) stages15 9-11. The embryo settles at the topmost part of the egg, so you should mark it with a pencil line.</li>
 <li>Disinfect the eggs with 70% ethanol. </li>
 <li>Pierce the egg at its broader side, where the air cavity should be. </li>
 <li>Remove 1-2 ml of egg white. This will detach the embryo from the eggshell. </li>
 <li>Cello-tape the eggshell to avoid shell splinters falling onto the embryo. Use a pair of scissors to cut a small window into the eggshell around the pencil line. </li>
 <li>Inject a bit of ink underneath the area opaca to visualize the embryo.</li>
 <li>Make a cut into the vitelline membrane with a sharpened tungsten needle to achieve a better accessibility of the embryo for nucleic acid injection and electroporation. </li>
 </ol>
 </li>
</ol>
3. Electroporation and Incubation
<ol>
 <li>Add a few drops of warm Ringer's solution onto the embryos. This will lower the embryos, prevent overheating, sticking of the electrodes to the tissue and provide a medium for the current between the two electrodes. </li>
 <li>Inject the vector solution into the lumen of the neural tube at midbrain level. If your DNA expression vector contains no reporter gene (e.g. EGFP), add a slightly lower concentrated reporter gene vector (1 μg/μl miR expressing vector to 0.9 μg/μl reporter gene vector) to the DNA solution. This will ensure that you recognize transfected cells later8. We use the so-called pCAX vector, a kind gift from C. Krull 13. </li>
 <li>For a broad transfection of the midbrain, place the electrodes left and right of the embryo at the height of the midbrain. The distance between the electrodes should be around 3-5 mm. The other settings are 15V, 20 msec pulses and 50 msec delay for HH 10. Changing the position of the two electrodes slightly along the dorso-ventral (DV) axis of the midbrain assures a more a broad transfection of cells the DV axis transfection. </li>
 <li>For ventral transfection, add some Ringer to raise the head from the yolk membrane and place the anode beneath ventral midbrain. If the head does not rise push the electrode under the membrane that separates yolk and embryo to avoid scorching of the neural tube. The cathode should be placed above the midbrain dorso-laterally opposite to the anode. Do not touch the neural tube. We transfect the left ventral half of the midbrain to avoid any difficulties with heart development. The distance between the electrodes is around 1-0.5 mm, the rest of the settings are 10V, 20 msec width and 50 msec delay.</li>
 <li>Apply another drop of Saline on top of the embryos to cool it down and remove air bubbles. </li>
 <li>Seal the eggs with the tape and incubate for at least 4 hr; the minimum time required for vector expression. </li>
</ol>
 4. Fixation of Chicken Embryos
 <ol>
 <li>Remove the embryos from their eggs. </li>
 <li>Clean embryos under a stereomicroscope and assess the extent of transfection with a fluorescent stereo microscope. </li>
 <li>Fix the embryos over night in 4% PFA at 4°C. Proceed with sectioning and/or staining (in situ hybridization, immunohistochemistry) to analyse changes in the transfected area. </li>
 </ol>

<ol><li>Kutter, C., Svoboda, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((miRNA%2C+siRNA%2C+piRNA%3A+Knowns+of+the+unknown%5BTitle%5D)+AND+%22RNA+Biol%22%5BJournal%5D)">miRNA, siRNA, piRNA: Knowns of the unknown.</a> RNA Biol. 5, 181-188 (2008).</li>
<li>Szell, M., Bata-Csorgo, Z., Kemeny, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+enigmatic+world+of+mRNA-like+ncRNAs%3A+their+role+in+human+evolution+and+in+human+diseases%5BTitle%5D)+AND+%22Semin.+Cancer+Biol%22%5BJournal%5D)">The enigmatic world of mRNA-like ncRNAs: their role in human evolution and in human diseases.</a> Semin. Cancer Biol. 18, 141-148 (2008).</li>
<li>Li, X., Jin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Roles+of+small+regulatory+RNAs+in+determining+neuronal+identity%5BTitle%5D)+AND+%22Nature+reviews.+Neuroscience%22%5BJournal%5D)">Roles of small regulatory RNAs in determining neuronal identity.</a> Nature reviews. Neuroscience. 11, 329-338 (2010).</li>
<li>Riedmann, L. T., Schwentner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((miRNA%2C+siRNA%2C+piRNA+and+argonautes%3A+news+in+small+matters%5BTitle%5D)+AND+%22RNA+Biol%22%5BJournal%5D)">miRNA, siRNA, piRNA and argonautes: news in small matters.</a> RNA Biol. 7, 133-139 (2010).</li>
<li>Coolen, M., Bally-Cuif, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MicroRNAs+in+brain+development+and+physiology%5BTitle%5D)+AND+%22Curr.+Opin.+Neurobiol%22%5BJournal%5D)">MicroRNAs in brain development and physiology.</a> Curr. Opin. Neurobiol. 19, 461-470 (2009).</li>
<li>Muramatsu, T., Mizutani, Y., Ohmori, Y., Okumura, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+three+nonviral+transfection+methods+for+foreign+gene+expression+in+early+chicken+embryos+in+ovo%5BTitle%5D)+AND+%22Biochem.+Biophys.+Res.+Commun%22%5BJournal%5D)">Comparison of three nonviral transfection methods for foreign gene expression in early chicken embryos in ovo.</a> Biochem. Biophys. Res. Commun. 230, 376-380 (1997).</li>
<li>Itasaki, N., Bel-Vialar, S., Krumlauf, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Shocking%27+developments+in+chick+embryology%3A+electroporation+and+in+ovo+gene+expression%5BTitle%5D)+AND+%22Nat.+Cell+Biol%22%5BJournal%5D)">Shocking' developments in chick embryology: electroporation and in ovo gene expression.</a> Nat. Cell Biol. 1, 203-207 (1999).</li>
<li>Momose, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+targeting+of+gene+expression+in+chick+embryos+by+microelectroporation%5BTitle%5D)+AND+%22Dev.+Growth+Differ%22%5BJournal%5D)">Efficient targeting of gene expression in chick embryos by microelectroporation.</a> Dev. Growth Differ. 41, 335-344 (1999).</li>
<li>Nakamura, H., Watanabe, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Misexpression+of+genes+in+brain+vesicles+by+in+ovo+electroporation%5BTitle%5D)+AND+%22Dev.+Growth+Differ%22%5BJournal%5D)">Misexpression of genes in brain vesicles by in ovo electroporation.</a> Dev. Growth Differ. 42, 199-201 (2000).</li>
<li>Voiculescu, O., Papanayotou, C., Stern, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spatially+and+temporally+controlled+electroporation+of+early+chick+embryos%5BTitle%5D)+AND+%22Nat.+Protoc%22%5BJournal%5D)">Spatially and temporally controlled electroporation of early chick embryos.</a> Nat. Protoc. 3, 419-426 (2008).</li>
<li>Neumann, E., Schaefer-Ridder, M., Wang, Y., Gene Hofschneider, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((transfer+into+mouse+lyoma+cells+by+electroporation+in+high+electric+fields%5BTitle%5D)+AND+%22The+EMBO+journal%22%5BJournal%5D)">transfer into mouse lyoma cells by electroporation in high electric fields.</a> The EMBO journal. 1, 841-845 (1982).</li>
<li>Potter, H., Weir, L., Leder, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enhancer-dependent+expression+of+human+kappa+immunoglobulin+genes+introduced+into+mouse+pre-B+lymphocytes+by+electroporation%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Enhancer-dependent expression of human kappa immunoglobulin genes introduced into mouse pre-B lymphocytes by electroporation.</a> Proceedings of the National Academy of Sciences of the United States of America. 81, 7161-7165 (1984).</li>
<li>Krull, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+primer+on+using+in+ovo+electroporation+to+analyze+gene+function%5BTitle%5D)+AND+%22Dev.+Dyn%22%5BJournal%5D)">A primer on using in ovo electroporation to analyze gene function.</a> Dev. Dyn. 229, 433-439 (2004).</li>
<li>Canto-Soler, M. V., Adler, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optic+cup+and+lens+development+requires+Pax6+expression+in+the+early+optic+vesicle+during+a+narrow+time+window%5BTitle%5D)+AND+%22Developmental+biology%22%5BJournal%5D)">Optic cup and lens development requires Pax6 expression in the early optic vesicle during a narrow time window.</a> Developmental biology. , 294-2119 (2006).</li>
<li>Hamburger, V., Hamilton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+series+of+normal+stages+in+the+development+of+the+chick+embryo.+1951%5BTitle%5D)+AND+%22Dev+Dyn%22%5BJournal%5D)">A series of normal stages in the development of the chick embryo. 1951.</a> Dev Dyn. 195, 231-272 (1992).</li>
<li>Muramatsu, T., Nakamura, A., Park, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+electroporation%3A+a+powerful+and+convenient+means+of+nonviral+gene+transfer+to+tissues+of+living+animals+%28Review%29%5BTitle%5D)+AND+%22Int+J+Mol+Med%22%5BJournal%5D)">In vivo electroporation: a powerful and convenient means of nonviral gene transfer to tissues of living animals (Review).</a> Int J Mol Med. 1, 55-62 (1998).</li>
<li>Agoston, Z., Li, N., Haslinger, A., Wizenmann, A., Schulte, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetic+and+physical+interaction+of+Meis2%2C+Pax3+and+Pax7+during+dorsal+midbrain+development%5BTitle%5D)+AND+%22BMC+Dev+Biol%22%5BJournal%5D)">Genetic and physical interaction of Meis2, Pax3 and Pax7 during dorsal midbrain development.</a> BMC Dev Biol. 12, 10(2012).</li>
<li>De Pietri Tonelli, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-cell+detection+of+microRNAs+in+developing+vertebrate+embryos+after+acute+administration+of+a+dual-fluorescence+reporter%2Fsensor+plasmid%5BTitle%5D)+AND+%22Biotechniques%22%5BJournal%5D)">Single-cell detection of microRNAs in developing vertebrate embryos after acute administration of a dual-fluorescence reporter/sensor plasmid.</a> Biotechniques. 41, 727-732 (2006).</li>
</ol>

We have nothing to disclose.

This video demonstrates an effective method to transfect plasmid into the neuroepithelial cells of specific areas of the chick midbrain. Rectangular electric pulses of low voltage can introduce DNA into cells of the chick neural tube in ovo6,16. However, the accuracy of DNA targeting is often hindered by the wide electric field, which rises through the relatively large electrodes (&Phi; = 0.5 mm). We tried to tackle that problem by using electrodes smaller in diameter following the guidelines of Momos...

The recognition of small non-coding RNAs as additional players for gene expression launched a new complexity to genomic programming/gene regulation. Different species of non-coding RNAs have functional importance in neural cells, including small non-coding RNAs1-4. MicroRNAs (miR or miRNA) for example show distinct and changing expression profiles in developing brains5. Targeted in ovo electroporation of chick embryos provides a unique opportunity for temporal and spatial control of gene expression and silencing during development. 
This video demonstrates the different steps of performing ectopic expression of miRs in specific areas of the chick midbrain using in ovo electroporation6-10. To ensure a long lasting effect of these small non-coding RNAs in cells, the DNA sequence of miRs were cloned into mono- or bi-cistronic vectors. For in ovo electroporation, miR containing vector is injected into the midbrain neural tube by exposing the embryo after making a small window in the egg shell. To transfect specific areas of the midbrain small plus (anode) and minus (cathode) platinum electrodes are placed at specific positions. For ventral midbrain transfection, the anode is placed underneath the left ventral midbrain and the cathode above the right half of the midbrain before applying a current. The opening in the eggshell is closed with tape and embryos are incubated for as long as required for any analysis. This method was originally described by Muramatsu et al.6 and improved by Momose et al.8 for specific area transfection.
<img src="/files/ftp_upload/50024/50024schematic1.jpg" fo:content-width-="6in" fo:src="/files/ftp_upload/50024/50024schematic1highres.jpg" alt="Gene editing process diagram; DNA→RNA; electrophoresis setup; molecular biology method." data-altupdatedat="1787630408000"> 
Schematic Overview. 
<ol>
 <li> The embryo in the egg is exposed by cutting a small window into the eggshell. </li>
 <li> The dissolved vector(s) is injected into the midbrain using a micro capillary. </li>
 <li>Two electrodes - placed parallel or under and above the embryo - generate a pulsed electric field.</li>
 <li> The electric field temporally creates pores in the cell membrane, which facilitate entry into the cell by the negatively charged DNA (or RNA) attracted to the anode11,12. </li>
</ol>

<table><tbody><tr><td>Borosillicate glass capillaries </td><td>Hartenstein</td><td></td><td>Model: 0.9 mm</td></tr><tr><td>Microcapillary puller</td><td>WPI, Berlin</td><td></td><td>Model: Pul1-E</td></tr><tr><td>Electroporator</td><td>Intracel</td><td></td><td>Model: TSSIC</td></tr><tr><td>Stereomicroscope - fluorescence </td><td>LEICA</td><td></td><td>Model: MZFLIII</td></tr><tr><td>Stereomicroscope</td><td>Zeiss</td><td></td><td>Model: Stemi</td></tr><tr><td>Camera and software</td><td>Zeiss</td><td></td><td>Model: Axiocam MRc/ Axiovision Re. 4.8</td></tr></tbody></table>

in ovo expression of microrna in ventral chick midbrain

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.

The extent of the midbrain area transfected with miR is visible through the expression of GFP from a reporter vector injected along with the miR expressing vector (Figure 2). Using platinum electrodes with a diameter of 0.5 mm and placed parallel to the AP axis of midbrain leads normally to the transfection of a broad area along the DV- and AP- axis, including hindbrain, midbrain and sometimes diencephalon (Figures 2A and B). The size of the electrodes results in a wide electric field ap...

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 ...

An oval electroporation begins with opening the act to expose the cheek embryo. At hamburger Hamilton, stages nine to 11 DNA is injected into the midbrain region. Then electrodes are placed left and right of the midbrain and an electrical voltage is applied.This causes a mural progenitor cells at the ventricular zone to take up the DNA For ventral Transfection. One electrode. The anode is positioned under the membrane covering the A below the midbrain area.The cathode is placed lateral dorsally at the opposite side of the midbrain before an electrical voltage is applied. Please take care that the electrodes do not contact the neural tube. 24 hours later at Hamburger Hamilton stage 16 or 17, the embryos are removed from the egg fixed and analyzed by antibodies, staining, or in situ hybridization.GFP positive cells indicate the area of gene transfect. Electroporation of microRNA precursors molecules, or a microRNA duplex provides an immediate but short effect to assure a longer expression of microRNAs. We electro operate an expression vector that contains sequences coding from micro RNA together with a vector expressing GFP.To visualize transected cells, MicroRNA expressed from a vec

in-ovo-expression-of-microrna-in-ventral-chick-midbrain

Research

JoVE Journal

Neuroscience

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Assay for Neural Induction in the Chick Embryo

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In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

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In ovo Electroporation of miRNA-based Plasmids in the Developing Neural Tube and Assessment of Phenotypes by DiI Injection in Open-book Preparations

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

Slice cultures facilitate the manipulation of embryo development by gene and pharmacological perturbations. However, culture conditions must ensure that normal development can proceed within the reduced environment of the slice. We illustrate a protocol that facilitates normal spinal cord development to proceed for at least 24 hr.

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Auditory brainstem neurons of avians and mammals are specialized for fast neural encoding, a fundamental process for normal hearing functions. These neurons arise from distinct precursors of embryonic hindbrain. We present techniques utilizing electroporation to express genes in the hindbrain of chicken embryos to study gene function during auditory development.

In Ovo Electroporation in the Chicken Auditory Brainstem

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.

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

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In Ovo and Ex Ovo Methods to Study Avian Inner Ear Development

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Dissecting Cell-Autonomous Function of Fragile X Mental Retardation Protein in an Auditory Circuit by In Ovo Electroporation

Learn in ovo electroporation for targeted microRNA expression in chick ventral midbrain. This method enables efficient DNA delivery to mural progenitor cells for studying brain development.

In ovo Expression of MicroRNA in Ventral Chick Midbrain

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