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 (&Phi; = 0.2, 0.25, or 0.5 mm in diameter) or in some cases an even smaller tungsten wire electrode (&Phi; = 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"/> 
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&deg;. 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 &mu;g/&mu;l 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 &deg;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 &mu;g/&mu;l miR expressing vector to 0.9 &mu;g/&mu;l 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&deg;C. Proceed with sectioning and/or staining (in situ hybridization, immunohistochemistry) to analyse changes in the transfected area. </li>
 </ol>

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Cell Biol. 1, 203-207 (1999).</li><li>Momose, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+targeting+of+gene+expression+in+chick+embryos+by+microelectroporation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Misexpression+of+genes+in+brain+vesicles+by+in+ovo+electroporation.">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. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+electroporation:+a+powerful+and+convenient+means+of+nonviral+gene+transfer+to+tissues+of+living+animals+(Review).">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+and+physical+interaction+of+Meis2,+Pax3+and+Pax7+during+dorsal+midbrain+development.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+detection+of+microRNAs+in+developing+vertebrate+embryos+after+acute+administration+of+a+dual-fluorescence+reporter/sensor+plasmid.">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"/> 
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

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...

Watch this Scientific Journal Video about In ovo Expression of MicroRNA in Ventral Chick Midbrain at JoVE.com

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.

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 ...

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

Research

JoVE Journal

Neuroscience

10.4K Views.  University of Tübingen.  Ectopische expressie is een techniek om de microRNA's rol in de ontwikkeling van de hersenen toe te lichten. Echter, gericht op specifieke gebieden met behulp van in-ovo elektroporatie is een uitdaging. Hier tonen we een efficiënte manier om selectief electroporate ventrale en dorsale middenhersenen regio. 

Video: In ovo Expression of MicroRNA in Ventral Chick Midbrain

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, cells from the roof of the blastocoel are pluripotent. These cells can be isolated, and programmed to generate various tissues through manipulation of genes expression or induction by morphogens. In this manuscript protocols are described for the use of Xenopus laevis blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development. These protocols allow the investigation of fate acquisition, cell migration behaviors, and cell autonomous and non-autonomous properties. The blastocoel roof explants can be cultured in a serum-free defined medium and grafted into host embryos. This transplantation into an embryo allows the investigation of the long-term lineage commitment, the inductive properties, and the behavior of transplanted cells in vivo. These assays can be exploited to investigate molecular mechanisms, cellular processes and gene regulatory networks underlying neural development. In the context of regenerative medicine, these assays provide a means to generate neural-derived cell types in vitro that could be used in drug screening.

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

In Xenopus embryos, cells from the roof of the blastocoel are pluripotent and can be programmed to generate various tissues. Here, we describe protocols to use amphibian blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development.

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, ...

Developmental Biology

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. The inner ear auditory sensory organ is critical for our ability to hear. It houses a highly specialized sensory epithelium that consists of mechano-transducing hair cells (HCs) and surrounding glial-like supporting cells (SCs). Despite structural differences in the auditory organs, molecular mechanisms regulating the development of the auditory organ are evolutionarily conserved between mammals and aves. In ovo electroporation is largely limited to early stages at E1 - E3. Due to the relative late development of the auditory organ at E5, manipulations of the auditory organ by in ovo electroporation past E3 are difficult due to the advanced development of the chicken embryo at later stages. The method presented here is a transient gene transfer method for targeting genes of interest at stage E4 - E4.5 in the developing chicken auditory sensory organ via in ovo micro-electroporation. This method is applicable for gain- and loss-of-functions with conventional plasmid DNA-based expression vectors and can be combined with in ovo cell proliferation assay by adding EdU (5-ethynyl-2&#180;-deoxyuridine) to the whole embryo at the time of electroporation. The use of green or red fluorescent protein (GFP or RFP) expression plasmids allows the experimenter to quickly determine whether the electroporation successfully targeted the auditory portion of the developing inner ear. In this method paper, representative examples of GFP electroporated specimens are illustrated; embryos were harvested 18 - 96 hr&#160;after electroporation and targeting of GFP to the pro-sensory area of the auditory organ was confirmed by RNA in situ hybridization. The method paper also provides an optimized protocol for the use of the thymidine analog EdU to analyze cell proliferation; an example of an EdU based cell proliferation assay that combines immuno-labeling and click EdU chemistry is provided.

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

The auditory organ mediates hearing. Here we present a modified in ovo micro-electroporation method optimized for studying auditory progenitor cell proliferation and differentiation in the developing chicken auditory organ.

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. ...

In Ovo Electroporation in the Chicken Auditory Brainstem

Electroporation is a method that introduces genes of interest into biologically relevant organisms like the chicken embryo. It is long established that the chicken embryo is an effective research model for studying basic biological functions of auditory system development. More recently, the chicken embryo has become particularly valuable in studying gene expression, regulation and function associated with hearing. In ovo electroporation can be used to target auditory brainstem regions responsible for highly specialized auditory functions. These regions include the chicken nucleus magnocellularis (NM) and nucleus laminaris (NL). NM and NL neurons arise from distinct precursors of rhombomeres 5 and 6 (R5/R6). Here, we present in ovo electroporation of plasmid-encoded genes to study gene-related properties in these regions. We show a method for spatial and temporal control of gene expression that promote either gain or loss of functional phenotypes. By targeting auditory neural progenitor regions associated with R5/R6, we show plasmid transfection in NM and NL. Temporal regulation of gene expression can be achieved by adopting a tet-on vector system. This is a drug inducible procedure that expresses the genes of interest in the presence of doxycycline (Dox). The in ovo electroporation technique &#8211; together with either biochemical, pharmacological, and or in vivo functional assays &#8211; provides an innovative approach to study auditory neuron development and associated pathophysiological phenomena.

In Ovo Electroporation in the Chicken Auditory Brainstem

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.

Electroporation is a method that introduces genes of interest into biologically relevant organisms like the chicken embryo. It is long established that ...

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 ...

Chicken Embryo Spinal Cord Slice Culture Protocol

Slice cultures can facilitate the manipulation of embryo development both pharmacologically and through gene manipulations. In this reduced system, potential lethal side effects due to systemic drug applications can be overcome. However, culture conditions must ensure that normal development proceeds within the reduced environment of the slice. We have focused on the development of the spinal cord, particularly that of spinal motor neurons. We systematically varied culture conditions of chicken embryo slices from the point at which most spinal motor neurons had been born. We assayed the number and type of motor neurons that survived during the culture period and the position of those motor neurons compared to that in vivo. We found that serum type and neurotrophic factors were required during the culture period and were able to keep motor neurons alive for at least 24 hr and allow those motor neurons to migrate to appropriate positions in the spinal cord. We present these culture conditions and the methodology of preparing the embryo slice cultures using eviscerated chicken embryos embedded in agarose and sliced using a vibratome.

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.

Slice cultures can facilitate the manipulation of  embryo development both pharmacologically and through gene manipulations. In  this reduced system, ...

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 ...

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing and adult tissue. An advance in this concept is Genetic Inducible Fate Mapping (GIFM), linking gene expression, cell fate, and cell behaviors in vivo, to create fate maps based on genetic lineage.
GIFM exploits X-CreER lines where X is a gene or set of gene regulatory elements that confers spatial expression of a modified bacteriophage protein, Cre recombinase (CreERT). CreERT contains a modified estrogen receptor ligand binding domain which renders CreERT sequestered in the cytoplasm in the absence of the drug tamoxifen. The binding of tamoxifen releases CreERT, which translocates to the nucleus and mediates recombination between DNA sequences flanked by loxP sites. In GIFM, recombination typically occurs between a loxP flanked Stop cassette preceding a reporter gene such as GFP.
Mice are bred to contain either a region- or cell type-specific CreER and a conditional reporter allele. Untreated mice will not have marking because the Stop cassette in the reporter prevents further transcription of the reporter gene. We administer tamoxifen by oral gavage to timed-pregnant females, which provides temporal control of CreERT release and subsequent translocation to the nucleus removing the Stop cassette from the reporter. Following recombination, the reporter allele is constitutively and heritably expressed. This series of events marks cells such that their genetic history is indelibly recorded. The recombined reporter thus serves as a high fidelity genetic lineage tracer that, once on, is uncoupled from the gene expression initially used to drive CreERT.
We apply GIFM in mouse to study normal development and ascertain the contribution of genetic lineages to adult cell types and tissues. We also use GIFM to follow cells on mutant genetic backgrounds to better understand complex phenotypes that mimic salient features of human genetic disorders.
This video article guides researchers through experimental methods to successfully apply GIFM. We demonstrate the method using our well characterized Wnt1-CreERT;mGFP mice by administering tamoxifen at embryonic day (E)8.5 via oral gavage followed by dissection at E12.5 and analysis by epifluorescence stereomicroscopy. We also demonstrate how to micro-dissect fate mapped domains for explant preparation or FACS analysis and dissect adult fate-mapped brains for whole mount fluorescent imaging. Collectively, these procedures allow researchers to address critical questions in developmental biology and disease models.

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Genetic Inducible Fate Mapping (GIFM) marks and tracks cells with fine spatial and temporal control in vivo and elucidates how cells from a specific genetic lineage contribute to developing and adult tissues. Demonstrated here are the techniques required to fate map E12.5 mouse embryos for epifluorescent and explant analysis.

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing ...

Biology

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

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

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

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

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

Fragile X mental retardation protein (FMRP) is an mRNA-binding protein that regulates local protein translation. FMRP loss or dysfunction leads to aberrant neuronal and synaptic activities in fragile X syndrome (FXS), which is characterized by intellectual disability, sensory abnormalities, and social communication problems. Studies of FMRP function and FXS pathogenesis have primarily been conducted with Fmr1 (the gene encoding FMRP) knockout in transgenic animals. Here we report an in vivo method for determining the cell-autonomous function of FMRP during the period of circuit assembly and synaptic formation using chicken embryos. This method employs stage-, site-, and direction-specific electroporation of a drug-inducible vector system containing Fmr1 small hairpin RNA (shRNA) and an EGFP reporter. With this method, we achieved selective FMRP knockdown in the auditory ganglion (AG) and in one of its brainstem targets, the nucleus magnocellularis (NM), thus providing a component-specific manipulation within the AG-NM circuit. Additionally, the mosaic pattern of the transfection allows within-animal controls and neighboring neuron/fiber comparisons for enhanced reliability and sensitivity in data analyzing. The inducible vector system provides temporal control of gene editing onset to minimize accumulating developmental effects. The combination of these strategies provides an innovative tool to dissect the cell-autonomous function of FMRP in synaptic and circuit development.

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

Using in ovo electroporation, we devised a method to selectively transfect the auditory inner ear and cochlear nucleus in chicken embryos to achieve a cell-group-specific knockdown of fragile X mental retardation protein during discrete periods of circuit assembly.

In ovo Expression of MicroRNA in Ventral Chick Midbrain

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