1. Remove eggs from the incubator
At E2-2.5 (~ St. 17 HH), remove the eggs and prepare them accordingly. They can be worked on immediately or returned to the incubator for a couple hours. The eggs can be placed at room temperature safely for over an hour. Eggs can stay out for the duration of the manipulations. Sometimes these can take an hour or more.
Note: The longer they sit at room temperature, the less likely they are to survive.
2. Preparation of India ink
<ol>
<li>Prepare a fresh solution of 4% India Ink in sterile PBS.</li>
<li>Fill a 3 mL syringe with the solution.</li>
<li>Place a 27 gauge, &frac12; inch needle on the syringe.</li>
<li>Using a pair of hemostats, bend the needle to a 90&deg; angle.</li>
</ol>
3. Opening the eggs and injection of India ink
<ol>
<li>Using a pair of forceps, peel back the tape and open the window, securing it by using the reverse side of the tape on the back side of the egg.</li>
<li>Guide in the angled needle until it lies just beneath the embryo. Placing the needle under the floating disk insures that the ink, which is toxic to the embryo, will not come in direct contact.</li>
<li>You need to only inject enough ink to create a comfortable contrast. This is typically less than &frac14; mL.</li>
</ol>
4. Opening the neural tube
<ol>
<li>Take a pair of fine dissection scissors and cut the thin membrane that lies on top, or sometimes around, the embryo.</li>
<li>Take a pair of 55 forceps or a tungsten probe and open the neural tube from posterior to anterior. Make sure the opening is large enough to place a bead into it.</li>
</ol>
Note: Depending on the age, a fine tungsten probe can be used to do the same thing.
5. Retrieving a coated heparin-acrylic bead
<ol>
<li>Using a pair of 55 forceps, find an intact bead in the solution. Let the bead attach itself to the tip, slowly close the forceps.</li>
<li>Remove the bead from the solution and bring it over on top of the egg.</li>
<li>Once the bead is out of its solution, it will not likely come off until it has been placed in the albumin.</li>
</ol>
6. Placing the bead
<ol>
<li>Once the bead has been placed on top of the target area, use a tungsten probe to guide it towards the forebrain or fix it in the hindbrain.</li>
<li>Add a few drops of PBS using a 10 &micro;L pipette.</li>
</ol>
7. Closing the egg
<ol>
<li>Close the egg using the thin piece of tape to shut the window.</li>
<li>Seal the egg according to the prepping protocol, available&nbsp;at <a href="https://www.jove.com/index/Details.stp?ID=306" target="_blank">https://www.jove.com/index/Details.stp?ID=306</a>.</li>
</ol>

<ol>
	<li>Korn, M. J., Cramer, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Windowing+Chicken+Eggs+for+Developmental+Studies.">Windowing Chicken Eggs for Developmental Studies.</a> Jounal of Visualized Experiments. 8, (2007).</li><li>Crossley, P. H., Martinez, S., Ohkubo, Y., Rubenstein, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordinate+expression+of+Fgf8,+Otx2,+Bmp4,+and+Shh+in+the+rostral+prosencephalon+during+development+of+the+telencephalic+and+optic+vesicles.">Coordinate expression of Fgf8, Otx2, Bmp4, and Shh in the rostral prosencephalon during development of the telencephalic and optic vesicles.</a> Neuroscience. 108, 183-206 (2001).</li><li>Alexandre, P., Bachy, I., Marcou, M., Wassef, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positive+and+negative+regulations+by+FGF8+contribute+to+midbrain+roof+plate+developmental+plasticity.">Positive and negative regulations by FGF8 contribute to midbrain roof plate developmental plasticity.</a> Development . 133, 2905-2913 (2006).</li></ol>

The authors have nothing to disclose.

Depending on the protein you wish to use, there are different protocols on how to prepare the beads. In these experiments, we use heparin-acrylic beads, but affi-gel beads can also be used. We have found that the heparin-acrylic beads are easier to manipulate in the aqueous environment of the egg albumin. Beads are soaked in 5 l of protein overnight at 4&deg;C. Control beads are placed in sterile PBS until the time of placement. The advantage in using an in ovo preparation is that the embryo is still alive and free to ...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Eggs</td>
<td>Animal</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Fertilized, E2-2.5 (~ St. 17 HH)
</td>
</tr>
<tr>
<td>Incubator</td>
<td>Profi-I</td>
<td>Lyon</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>India Ink</td>
<td>Tool</td>
<td>Higgins</td>
<td>&nbsp;</td>
<td>Prepare a 4% solution in sterile PBS.</td>
</tr>
<tr>
<td>Sterile PBS</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Alternatively, an antibody cocktail can be used</td>
</tr>
<tr>
<td>1ml syringe</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>27G 1/2 needles</td>
<td>Tool</td>
<td>BD</td>
<td>305109</td>
<td>Angled at 90 degrees</td>
</tr>
<tr>
<td>Forceps</td>
<td>Tool</td>
<td>World Precision Instruments</td>
<td>&nbsp;</td>
<td>size 55</td>
</tr>
<tr>
<td>Hemostats</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Dissection scissors</td>
<td>Tool</td>
<td>World Precision Instruments</td>
<td>500086</td>
<td>vannas, 8.5cm</td>
</tr>
<tr>
<td>Parafilm tape</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>to seal eggs</td>
</tr>
<tr>
<td>Tungsten Probe</td>
<td>Tool</td>
<td>Electron Microscopy Sciences</td>
<td>62091</td>
<td>Tool #24. Handle optional. </td>
</tr>		</tbody>
		</table>
		</div>
			<div>
					</div>

placing growth factor-coated beads on early stage chicken embryos

The neural tube expresses many proteins in specific spatiotemporal patterns during development. These proteins have been shown to be critical for cell fate determination, cell migration, and formation of neural circuits. Neuronal induction and patterning involve bone morphogenetic protein (BMP), sonic hedgehog (SHH), fibroblast growth factor (FGF), among others. In particular, the expression pattern of Fgf8 is in close proximity to regions expressing BMP4 and SHH. This expression pattern is consistent with developmental interactions that facilitate patterning in the telencephalon.

Here we provide a visual demonstration of a method in which an in ovo preparation can be used to test the effects of Fgfs in the formation of the forebrain. Beads are coated with protein and placed in the developing neural tube to provide sustained exposure. Because the procedure uses small, carefully placed beads, it is minimally invasive and allows several beads to be placed within a single neural tube. Moreover, the method allows for continued development so that embryos can be analyzed at a more mature stage to detect changes in anatomy and in neural patterning. This simple but useful protocol allows for real time imaging. It provides a means to make spatially and temporally limited changes to endogenous protein levels.

Watch this Scientific Journal Video about Placing Growth Factor-Coated Beads on Early Stage Chicken Embryos at JoVE.com

Placing Growth Factor-Coated Beads on Early Stage Chicken Embryos

A variety of growth factors and proteins interact to induce cells to take on different cell fates during development. Here we demonstrate the use of an in ovo preparation to address possible interactions between different proteins in development by placing beads on E2.5 chick embryos.

The neural tube expresses many proteins in specific spatiotemporal patterns during development.   These proteins have been shown to be critical for cell ...

placing-growth-factor-coated-beads-on-early-stage-chicken-embryos

Research

JoVE Journal

Biology

11.4K ビュー.  University of California, Irvine (UCI). 成長因子や様々なタンパク質は、開発中に別の細胞の運命を引き受ける細胞を誘導するために相互作用する。ここでは、E2.5のニワトリ胚にビーズを配置することにより、開発の異なるタンパク質間の相互作用の可能性に対処するためにOVO準備での使用を示します。

ビデオ: Placing Growth Factor-Coated Beads on Early Stage Chicken Embryos

Growth Factor-Coated Bead Placement on Dorsal Forebrain Explants

This video demonstrates two methods for preparing and placing beads, which have been coated with growth factor, on explants of the developing cerebral cortex. These beads can be used to induce spatially restricted gene expression on developing neural tissue such as forebrain explants. Methods are given for using both Affi-Gel beads and heparin acryllic beads.

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos and their dechorionation using bleach are demonstrated. Using a glass pipet attached to a mouth suction tube, we illustrate the removal of all cells from single embryos. The method for dispersing cells from each embyro into a small (5 l) drop of medium on an uncoated glass coverslip is demonstrated. A view through the microscope at 1 hour after plating illustrates the preferred cell density. Most of the cells that survive when grown in defined medium are neuroblasts that divide one or more times in culture before extending neuritic processes by 12-24 hours. A view through the microscope illustrates the level of neurite outgrowth and branching expected in a healthy culture at 2 days in vitro. The cultures are grown in a simple bicarbonate based defined medium, in a 5% CO2 incubator at 22-24&deg;C. Neuritic processes continue to elaborate over the first week in culture and when they make contact with neurites from neighboring cells they often form functional synaptic connections. Neurons in these cultures express voltage-gated sodium, calcium, and potassium channels and are electrically excitable. This culture system is useful for studying molecular genetic and environmental factors that regulate neuronal differentiation, excitability, and synapse formation/function.

This video demonstrates the preparation of primary neuronal cultures from midgastrula stage Drosophila embryos. Views of live cultures show cells 1 hour after plating and differentiated neurons after 2 days of growth in a bicarbonate-based defined medium. The neurons are electrically excitable and form synaptic connections.

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos ...

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 Culture of Early Chick Embryos ex vivo (New Culture)

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 patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

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

Double Whole Mount in situ Hybridization of Early Chick Embryos

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  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo. Here we present a method to perform ChIP using a dissociated embryonic day 8.5 (E8.5) embryo. Sheared chromatin from a single E8.5 embryo can be divided into up to five aliquots, which allows the investigator sufficient material for controls and for investigation of specific protein:chromatin interactions.
We have utilized this technique to begin to document protein:chromatin interactions during the specification of tissue-specific gene expression programs. The heterogeneity of cell types in an embryo necessarily restricts the application of this technique because the result is the detection of protein:chromatin interactions without distinguishing whether the interactions occur in all, a subset of, or a single cell type(s). However, examination of tissue-specific genes during or following the onset of tissue-specific gene expression is feasible for two reasons. First, immunoprecipitation of tissue specific factors necessarily isolates chromatin from the cell type where the factor is expressed. Second, immunoprecipitation of coactivators and histones containing post-translational modifications that are associated with gene activation should only be found at genes and gene regulatory sequences in the cell type where the gene is being or has been activated. The technique should be applicable to the study of most tissue-specific gene activation events.
In the example described below, we utilized E8.5 and E9.5 mouse embryos to examine factor binding at a skeletal muscle specific gene promoter. Somites, which are the precursor tissues from which the skeletal muscles of the trunk and limbs will form, are present at E8.5-9.54,5. Myogenin is a regulatory factor required for skeletal muscle differentiation 6-9. The data demonstrate that myogenin is associated with its own promoter in E8.5 and E9.5 embryos. Because myogenin is only expressed in somites at this stage of development 6,10, the data indicate that myogenin interactions with its own promoter have already occurred in skeletal muscle precursor cells in E8.5 embryos.

We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development.

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This ...

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

The chicken embryo provides an excellent model system for studying gene function and regulation during embryonic development. In ovo electroporation is a powerful method to over-express exogenous genes or down-regulate endogenous genes in vivo in chicken embryos1. Different structures such as DNA plasmids encoding genes2-4, small interfering RNA (siRNA) plasmids5, small synthetic RNA oligos6, and morpholino antisense oligonucleotides7 can be easily transfected into chicken embryos by electroporation. However, the application of in ovo electroporation is limited to embryos at early incubation stages (younger than stage HH20 - according to Hamburg and Hamilton)8 and there are some disadvantages for its application in embryos at later stages (older than stage HH22 - approximately 3.5 days of development). For example, the vitelline membrane at later stages is usually stuck to the shall membrane and opening a window in the shell causes rupture of the vessels, resulting in death of the embryos; older embryos are covered by vitelline and allantoic vessels, where it is difficult to access and manipulate the embryos; older embryos move vigorously and is difficult to control the orientation through a relatively small window in the shell.
In this protocol we demonstrate an ex ovo electroporation method for gene transfer into chicken embryos at late stages (older than stage HH22). For ex ovo electroporation, embryos are cultured in Petri dishes9 and the vitelline and allantoic vessels are widely spread. Under these conditions, the older chicken embryos are easily accessed and manipulated. Therefore, this method overcomes the disadvantages of in ovo electroporation applied to the older chicken embryos. Using this method, plasmids can be easily transfected into different parts of the older chicken embryos10-12.

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

A method of gene transfer into chicken embryos at later incubation stages (older than Hamburger and Hamilton stage (HH) 22) is described. This method overcomes disadvantages of in ovo electroporation applied to older chicken embryos and is a useful technique to study gene function and regulation at older developmental stages.

The chicken embryo provides an  excellent model system for studying gene function and regulation during  embryonic development. In ovo electroporation is ...

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays.

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

We report an efficient and simple method to isolate embryos at early stages of development from Arabidopsis thaliana seeds. Up to 40 embryos can be isolated in 1 hr to 4 hr, depending on the downstream application. The procedure is suitable for transcriptome, DNA methylation, reporter gene expression, immunostaining and fluorescence in situ hybridization analyses.

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a ...

Dissection and Downstream Analysis of Zebra Finch Embryos at Early Stages of Development

The zebra finch (Taeniopygiaguttata) has become an increasingly important model organism in many areas of research including toxicology1,2, behavior3, and memory and learning4,5,6. As the only songbird with a sequenced genome, the zebra finch has great potential for use in developmental studies; however, the early stages of zebra finch development have not been well studied. Lack of research in zebra finch development can be attributed to the difficulty of dissecting the small egg and embryo. The following dissection method minimizes embryonic tissue damage, which allows for investigation of morphology and gene expression at all stages of embryonic development. This permits both bright field and fluorescence quality imaging of embryos, use in molecular procedures such as in situ hybridization (ISH), cell proliferation assays, and RNA extraction for quantitative assays such as quantitative real-time PCR (qtRT-PCR).&#160;This technique allows investigators to study early stages of development that were previously difficult to access.

The zebra finch (Taeniopygiaguttata) is a valuable model organism; however, early stages of zebra finch development have not been extensively studied. The protocol describes how to dissect early embryos for developmental and molecular applications.

The zebra finch (Taeniopygiaguttata) has become an increasingly important model organism in many areas of research including toxicology1,2, behavior3, and ...

Medium-throughput Screening Assays for Assessment of Effects on Ca2+-Signaling and Acrosome Reaction in Human Sperm

Ca2+-signaling is essential to normal sperm cell function and male fertility. Similarly, the acrosome reaction is vital for the ability of a human sperm cell to penetrate the zona pellucida and fertilize the egg. It is therefore of great interest to test compounds (e.g., environmental chemicals or drug candidates) for their effect on Ca2+-signaling and acrosome reaction in human sperm either to examine the potential adverse effects on human sperm cell function or to investigate a possible role as a contraceptive. Here, two medium-throughput assays are described: 1) a fluorescence-based assay for assessment of effects on Ca2+-signaling in human sperm, and 2) an image cytometric assay for assessment of acrosome reaction in human sperm. These assays can be used to screen a large number of compounds for effects on Ca2+-signaling and acrosome reaction in human sperm. Furthermore, the assays can be used to generate highly specific dose-response curves of individual compounds, determine potential additivity/synergism for two or more compounds, and to study the pharmacological mode of action through competitive inhibition experiments with CatSper inhibitors.

Medium-throughput Screening Assays for Assessment of Effects on Ca2+-Signaling and Acrosome Reaction in Human Sperm

Here, two medium-throughput assays for assessment of effects on Ca2+-signaling and acrosome reaction in human sperm are described. These assays can be used to quickly and easily screen large amounts of compounds for effects on Ca2+-signaling and acrosome reaction in human sperm.

Ca2+-signaling is essential to normal sperm cell function and male fertility. Similarly, the acrosome reaction is vital for the ability of a human sperm ...