We would like to thank Mr. Maaz Enver for the use of his HD camera (Cannon VIXIA HFS100). This work was supported in part by grants from NIH (EY018738), New Jersey Commission on Spinal Cord Research (08-3074-SCR-E-0 and 10-3091-SCR-E-0), and Busch Biomedical Research Award.

Since some parts of the protocol are performed as previously described in other JoVE videos2 and papers3 with minor modification we will not discuss those in detail here.
1. Egg Handling and Needle Preparation
<ol>
 <li>Eggs can be stored in a wine cooler at about 13&deg;C for up to 1 week. If the temperature is too high, embryos will start to develop abnormally, while lower temperature causes high mortality. Once you are ready to incubate take out the eggs from the wine cooler and set the eggs vertically with the larger end up. Keep the eggs in the room temperature for at least 2 hours before putting them in the 37.5&deg;C incubator. </li>
 <li>Incubate the eggs for 96-100 hours which is about embryonic day 4 to obtain embryos that are at the Hamburger and Hamilton stage 224-5. </li>
 <li>Prepare micropipette needles from pulled glass capillary tubes and break the tip under a dissecting microscope with a tweezers to get a tip opening about 0.1 &mu;m in diameter and a 20 mm taper. Needles with larger tips have difficulty piercing the vitelline membrane while smaller tips have difficulty loading and delivering the DNA solution. </li>
 <li>Attach the needle to a 0.1 ml Hamilton Gastight syringe mounted on a micromanipulator. Use a small piece of Masterplex silicone tube for attaching the needle to the syringe. Since the connections are tight no need to add mineral oil to seal this attachment. </li>
 <li>Mix 2 &mu;l of reporter plasmid DNA solution with a concentration ranging 3-6 &mu;g/&mu;l and 0.2 &mu;l of fast green (0.025%) on a piece of parafilm. Fast green dye will help to visualize the injection. Slowly, load the needle with the mixture. </li>
 <li>To free the vitelline membrane from the inner membrane rotate the egg gently about 180&deg; and wait for few minutes then rotate it back to original position and set it for electroporation. </li>
 <li>Wipe the forceps and egg shell with 70 % ethanol to avoid infection to the embryo. </li>
 <li>Make a small hole on the egg immediately above the air cell with a pair of size AA forceps. Be careful not to crack the egg shell. Remove small pieces one at a time to make a small window. Carefully remove the inner membrane using the forceps without touching the vitelline membrane. </li>
</ol>
2. Injection and Electroporation
<ol>
 <li>To prevent damage to the brain or the heart, position the needle contra lateral to the main bundle of blood vessels entering the eye and pointing towards the beak. </li>
 <li>Pierce through the vitelline membrane, sclera, retina and vitreous humor by a sudden mild push of the needle. If the needle pierces through the other end of the eye it should be alright unless it damages any major blood vein. </li>
 <li>Slowly pull back the needle at the edge of the opening and place it almost tangent to the outer wall of the eyeball. </li>
 <li>Insert the needle into the sub retinal space between the sclera and retina. </li>
 <li>Inject the DNA until you can visualize the green solution filling the side of the eyeball and pushing the retina inwards by creating a bulge. </li>
 <li>If your needle placing is not correct then you will see the DNA solution spreading inside the vitreous humor filling up the middle of the eye ball. Also, if you damage the retina too much then you will see the DNA solution is coming out of the eyeball. </li>
 <li>Slowly remove the needle and immediately place the electrodes in parallel inside the egg after soaking in PBS. Push down the electrodes to submerge into the amniotic fluid in a way so that the injected eye is located between the electrodes. Avoid touching any major blood vessel or the heart with the electrodes while placing them. The negative electrode should be at the injection side so that DNA can be transported from sub retinal space into the retina towards positive electrode. Electroporate the retina with 5 pulses of 15V for 50 ms with 950 ms intervals.</li>
 <li>Carefully remove the electrodes and seal the window of the egg with pieces of clear scotch tape. </li>
 <li>Label and date the injected egg before putting it back to the incubator. Typically, it takes about 3 to 5 minutes to complete the whole electroporation process. </li>
 <li>GFP expression can be seen as early as 8 hours after electroporation. However, you may wait until the embryo reaches the desired stage before harvesting. </li>
</ol>
3. Representative Results
In our study, we use various plasmid constructs to study the regulation of gene expression that involved retinal cell development. In this video pCAG-GFP (transfection control) was used to follow a successful injection and electroporation. However, any plasmid construct with reporter gene (GFP, RFP etc.) can be used. Even though GFP expression can be seen as early as 8 hours after electroporation, we typically start harvesting the egg on day 6 (E6) and onwards. Electroporated retinas were dissected out of the embryo and analyzed under fluorescent dissection microscope before embedding and sectioning. Typically, reporter gene expression can be seen at least in a quarter of the retina after a successful electroporation (Fig 1). The transfected retinal tissues were further analyzed through sectioning for clear visualization of cell morphologies. Immunohistochemistry using cell type specific markers (Brn3a, Pax6 etc.) allowed characterization of cell-specific GFP expression (Fig 2). 
<img src="/files/ftp_upload/3792/3792fig1.jpg" align="Figure 1" /> 
Figure 1. Successful electroporation of reporter plasmid results positive GFP expression. Chicken embryonic retinas were injected and electroporated at embryonic day 4 and harvested at embryonic day 6. At least 25% of the retina was successfully transfected (A=Top view, B= Bottom view). Scale Bar = 1mm.
<img src="/files/ftp_upload/3792/3792fig2.jpg" align="Figure 2" /> 
Figure 2. Characterization of GFP expressing retinal cells using immunohistochemistry. GFP expressing retinal tissues at E7 stage were fixed and sectioned. These sections were then stained with various cell specific markers. Brn3a was used to determine ganglion cells (A) while Pax6 was used to determine horizontal, amacrine and ganglion cells (B). Scale bar = 50 &mu;m.

<ol>
	<li>Doh, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+retinal+cell+development+in+chick+embryo+by+immunohistochemistry+and+in+ovo+electroporation+techniques.">Analysis of retinal cell development in chick embryo by immunohistochemistry and in ovo electroporation techniques.</a> BMC. Dev. Biol. 10, (2010).</li><li>Blank, M. C., Chizhikov, V., Millen, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Ovo+Electroporations+of+HH+Stage+10+Chicken+Embryos.">In Ovo Electroporations of HH Stage 10 Chicken Embryos.</a> J. Vis. Exp. (9), e408-e408 (2007).</li><li>Nakamura, H., Funahashi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+of+DNA+into+chick+embryos+by+in+ovo+electroporation.">Introduction of DNA into chick embryos by in ovo electroporation.</a> Methods. 24, 43-48 (2001).</li><li>Hamburger, V., Hamilton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+series+of+normal+stages+in+the+development+of+the+chick+embryo.+1951.">A series of normal stages in the development of the chick embryo. 1951.</a> Dev. Dyn.. 195, 231-272 (1992).</li><li>Hamburger, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+stage+series+of+the+chick+embryo.">The stage series of the chick embryo.</a> Dev. Dyn. 195, 273-275 (1992).</li></ol>

All of the animal experiments were approved by the Institutional Animal Care and Facilities Committee at Rutgers University.

Targeted retinal injection and in ovo electroporation in embryonic retina at E4 stage can specifically target retinal progenitor cells resulting in the ability to visualize all six major retina cell types at the single cell level. In ovo electroporation at HH10 (~E1.5) targeting the optic vesicle is able to transfect cells that develop to form the eye. However, these cells have a very high turnover at this time and this method is not specific for retinal cells. It may be that the high cell turnover rate...

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Fertilized pathogen-free (SPF) white leghorn 			chicken eggs</td>
 <td>Sunrise 			Farms (Catskill, NY)</td>
 <td>&nbsp;
 </td>
 <td>&nbsp;
 </td>
 </tr>
 <tr>
 <td>Chicken egg incubator</td>
 <td>GQF 			manufacturing, Savannah, GA</td>
 <td>Model-1550
 </td>
 <td>Set to 60% humidity and 37.5&deg;C.</td>
 </tr>
 <tr>
 <td>Glass capillary tubes</td>
 <td>World 			Precision Instrument Inc.</td>
 <td>TW150F-4</td>
 <td>&nbsp;
 </td>
 </tr>
 <tr>
 <td>0.1 ml syringe</td>
 <td>Hamilton 			Co. Reno, Nevada</td>
 <td>Gastight 			1710</td>
 <td>Alternatively 1ml syringe can be used as well</td>
 </tr>
 <tr>
 <td>Masterflex Silicone (peroxide) Tubing</td>
 <td>Cole-Parmer</td>
 <td>HV-96400-13</td>
 <td>Cut a small piece (1 cm) for attaching the glass 			needle to the syringe</td>
 </tr>
 <tr>
 <td>Tweezers</td>
 <td>Dumont</td>
 <td>AA</td>
 <td>&nbsp;
 </td>
 </tr>
 <tr>
 <td>Micromanipulator</td>
 <td>World 			Precision Instrument Inc.</td>
 <td>M3301-M3</td>
 <td>&nbsp;
 </td>
 </tr>
 <tr>
 <td>Plasmid DNA</td>
 <td>&nbsp;
 </td>
 <td>&nbsp;
 </td>
 <td>pCAG-GFP was borrowed from Dr. Connie Cepko</td>
 </tr>
 <tr>
 <td>Fast Green FCF</td>
 <td>Sigma-Aldrich</td>
 <td>F7252</td>
 <td>Dilute it to 0.025 % with PBS</td>
 </tr>
 <tr>
 <td>Pulse generator</td>
 <td>Harvard 			Apparatus, MA</td>
 <td>BTX 			ECM 830</td>
 <td>Square wave generator</td>
 </tr>
 <tr>
 <td>Electrodes</td>
 <td>Harvard Apparatus, MA</td>
 <td>BTX 			model 514</td>
 <td>Our electrodes were spaced 3-5 mm apart</td>
 </tr>
 <tr>
 <td>Monochrome Digital Camera</td>
 <td>Zeiss, Germany</td>
 <td>Axiocam 			MRM</td>
 <td>&nbsp;
 </td>
 </tr>
 <tr>
 <td>Fluorescent Dissection Microscope</td>
 <td>Leica Microsystems, Germany</td>
 <td>Leica 			MZ16FA</td>
 <td>&nbsp;
 </td>
 </tr>
 <tr>
 <td>Upright Fluorescence
 Microscope</td>
 <td>Zeiss, Germany</td>
 <td>Zeiss 			Axio Imager A1</td>
 <td>&nbsp;
 </td>
 </tr>
 </tbody>
</table>

in ovo electroporation in embryonic chick retina

Chicken embryonic retina is an excellent tool to study retinal development in higher vertebrates. Because of large size and external development, it is comparatively very easy to manipulate the chick embryonic retina using recombinant DNA/RNA technology. Electroporation of DNA/RNA constructs into the embryonic retina have a great advantage to study gene regulation in retinal stem/progenitor cells during retinal development. Different type of assays such as reporter gene assay, gene over-expression, gene knock down (shRNA) etc. can be performed using the electroporation technique. This video demonstrates targeted retinal injection and in ovo electroporation into the embryonic chick retina at the Hamburger and Hamilton stage 22-23, which is about embryonic day 4 (E4). Here we show a rapid and convenient in ovo electroporation technique whereby a plasmid DNA that expresses green fluorescent protein (GFP) as a marker is directly delivered into the chick embryonic subretinal space and followed by electric pulses to facilitate DNA uptake by retinal stem/progenitor cells. The new method of retinal injection and electroporation at E4 allows the visualization of all retinal cell types, including the late-born neurons1, which has been difficult with the conventional method of injection and electroporation at E1.52.

Watch this Scientific Journal Video about In Ovo Electroporation in Embryonic Chick Retina at JoVE.com

In Ovo Electroporation in Embryonic Chick Retina

The overall goal of this video is to show how to perform targeted retinal injection and in ovo electroporation of DNA/RNA constructs into the chick embryonic retina at the Hamburger and Hamilton stage 22-23, which is about embryonic day 4 (E4). This technique is very useful to study gene expression, gene regulation, and morphological change in developing chick retina.

In Ovo Electroporation in Embryonic Chick Retina

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

in-ovo-electroporation-in-embryonic-chick-retina

Research

JoVE Journal

Biology

14.3K 次观看.  University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School. 该视频的总体目标是显示如何执行有针对性的视网膜注射在OVO DNA / RNA的结构，在汉堡包和汉密尔顿22-23阶段，这是关于胚胎第4天（E4），成鸡胚胎视网膜电。这种技术是非常有用的，以研究基因表达，基因调控，并在发展中国家小鸡视网膜的形态学变化。

视频: In Ovo Electroporation in Embryonic Chick Retina

In Ovo Electroporations of HH Stage 10 Chicken Embryos

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

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

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

Method for Whole Mount Antibody Staining in Chick

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Assay for Neural Induction in the Chick Embryo

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying the formation and initial patterning of the nervous system. This video demonstrates how to graft organizer tissue into a host, a method by which Hensen s node (the organizer in the chick embryo) is grafted to a host competent ectoderm. The organizer graft instructs overlying na ve tissue to adopt a neural fate via neural inducing signals. This mechanism is referred to as neural induction, and constitutes the initial step in the formation of brain and spinal cord in amniotes. This method is essentially used for the characterization of putative neural inducing molecules in chick. This video demonstrates the different steps in the assay for neural induction; First, the donnor embryo is explanted and pinned on a dish. Then, the host embryo is prepared for New culture. The graft is excised and transplanted to the host area pellucida margin. The host is cultured for 18-22 hrs. The assembly is fixed and processed for further applications (e.g. in situ hybridization). This method was originally devised by Waddington 1,2 and Gallera 3,4.

Neural induction is the first step in the formation of the brain. It is a mechanism by which Hensen's node (organizer), instructs adjacent tissue to adopt a neural fate, i.e. to give rise to the nervous system. This video demonstrates an assay for neural induction in chick embryo.

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study angiogenesis but also to study tumorigenesis. After the chick chorioallantoic membrane (CAM) has developed, its blood vessel network can be easily accessed, manipulated and observed and therefore provides an optimal setting for angiogenesis assays. Since the lymphoid system is not fully developed until late stages of incubation, the chick embryo serves as a naturally immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition to nurturing developing allo- and xenografts, the CAM blood vessel network provides a uniquely supportive environment for tumor cell intravasation, dissemination, and vascular arrest and a repository where arrested cells extravasate to form micro metastatic foci.
For experimental purposes, in most of the recent studies the CAM was exposed by cutting a window through the egg shell and experiments were carried out in ovo, resulting in significant limitations in the accessibility of the CAM and possibilities for observation and photo documentation of effects. When shell-less cultures of the chick embryo were used1-4, no experimental details were provided and, if published at all, the survival rates of these cultures were low. We refined the method of ex ovo culture of chick embryos significantly by introducing a rationally controlled extrusion of the egg content. These ex ovo cultures enhance the accessibility of the CAM and chick embryo, enabling easy in vivo documentation of effects and facilitating experimental manipulation of the embryo. This allows the successful application to a large number of scientific questions: (1) As an improved angiogenesis assay5,6, (2) an experimental set up for facilitated injections in the vitreous of the chick embryo eye7-9, (3) as a test environment for dissemination and intravasation of dispersed tumor cells from established cell lines inoculated on the CAM10-12, (4) as an improved sustaining system for successful transplantation and culture of limb buds of chicken and mice13 as well as (5) for grafting, propagation, and re-grafting of solid primary tumor tissue obtained from biopsies on the surface of the CAM14.
In this video article we describe the establishment of a refined chick ex ovo culture and CAM assay with survival rates over 50%. Besides we provide a step by step demonstration of the successful application of the ex ovo culture for a large number of scientific applications. 
Daniel S. Dohle, Susanne D. Pasa, and Sebastian Gustmann contributed equally to this study.

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

The chick chorioallantoic membrane (CAM) is a unique, naturally immunodeficient supportive culture environment to study angiogenesis and tumorigenesis. This video article demonstrates the different steps in chick ex ovo culture, application of potentially angiogenic substances and successful inoculation of tumor cells and tissues on the surface of the CAM.

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study ...

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation

Employment of enhancer elements to drive expression of reporter genes in neurons is a widely used paradigm for tracking axonal projection. For tracking axonal projection of spinal interneurons in vertebrates, germ line-targeted reporter genes yield bilaterally symmetric labeling. Therefore, it is hard to distinguish between the ipsi- and contra-laterally projecting axons. Unilateral electroporation into the chick neural tube provides a useful means to restrict expression of a reporter gene to one side of the central nervous system, and to follow axonal projection on both sides 1 ,2-5. This video demonstrates first how to handle the eggs prior to injection. At HH stage 18-20, DNA is injected into the sacral level of the neural tube, then tungsten electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator. The egg is sealed with tape and placed back into an incubator for further development. Three days later (E6) the spinal cord is removed as an open book preparation from embryo, fixed, and processed for whole mount antibody staining. The stained spinal cord is mounted on slide and visualized using confocal microscopy.

Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation

This video demonstrates how to visualize axonal pathways of genetically defined groups of neurons in the embryonic chick spinal cord utilizing in ovo electroporation of reporter genes under the control of specific enhancer elements.

Employment of enhancer elements to drive expression of reporter genes in neurons is a widely used paradigm for tracking axonal projection. For tracking ...

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

Single Cell Transfection in Chick Embryos

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a thorough analysis of the fates of single cells under normal and experimental conditions. To this end, transfection methods that target single progenitors are a prerequisite. We describe here a technically straightforward method for transfecting single cells in chicken tissues in-ovo, allowing reliable lineage tracing as well as genetic manipulation. Specific tissue domains are targeted within the somite or neural tube, and DNA is injected directly into the epithelium of interest, resulting in sporadic transfection of single cells. Using reporters, clonal populations may consequently be traced for up to three days, and behavior of genetically manipulated clonal populations can be compared with that of controls. This method takes advantage of the accessibility of the chick embryo along with emerging tools for genetic manipulation. We compare and discuss its advantages over the widely-used electroporation method, and possible applications and use in additional in-vivo models are also suggested. We advocate the use of this method as a significant addition and complement for existing lineage tracing and genetic interference tools.

Using fine tip micropipettes we inject plasmid DNA into subdomains of chicken somites or neural tubes. The concentration of the plasmid is adjusted to generate single transfected cells. We then allow the cells to develop into clonal populations.

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

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

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

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

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