Name: windowing chicken eggs for developmental studies
Uploaded: 2007-10-01T00:00:00
Description: Watch this Scientific Journal Video about Windowing Chicken Eggs for Developmental Studies at JoVE.com

1。インキュベーターから卵を取り除く<ol><li> 37維持° C 60％以上に設定された相対湿度と。 </li><li>卵を取り外し、卵を90 °回転、大きな基盤が水平に横たわるように。 </li></ol> 2。綿棒は、卵消毒に<ol><li> 70％エタノールで非滅菌ガーゼのスタックを飽和させる。 </li><li> 5卵にまで綿棒に2〜3個を使用してください。ガーゼが汚れた場合は破棄する。 </li></ol> 3...

<ol>
	<li>Cramer, K. S., Fraser, S. E., Rubel, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Dev Biol. 224, 138-151 (2000).</li><li>Cramer, K. S., Bermingham-McDonogh, O., Krull, C. E., Rubel, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Dev Biol. 269, 26-35 (2004).</li><li>Cramer, K. S., Cerretti, D. P., Siddiqui, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Dev Biol. 295, 76-89 (2006).</li><li>Huffman, K. 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=.">.</a> Dev Neurobiol. 67, 1655-1668 (2007).</li><li>Krull, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Dev Dyn. 229, 433-439 (2004).</li><li>Gerlach-Bank, L. M., Cleveland, A. R., Barald, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Dev Dyn. 229, 219-230 (2004).</li></ol>

このメソッドは、実験操作後の長期生存時間が可能になります。例えば、色素標識研究は、後に運命のマッピングの研究（1）E10またはに生存を許さ。さらに、それは後の発達段階（2-6）に進むために遺伝子の発現レベルと必要な開発の変化を提供するOVOエレクトロポレーション研究のために使用されています。このウィンドウテクニックは、胚への操作後の生存を必要とするすべての手順と組み合わせて使...

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<td>Rotating incubator</td>
<td>&nbsp;</td>
<td>Profi</td>
<td>&nbsp;</td>
<td>Maintain at 37&#176; Degrees with relative humidity set above 60%</td>
</tr>
<tr>
<td>70 % EtOH</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>gauze</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>3M plastic tape</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>10cc syringe</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>1/egg
</td>
</tr>
<tr>
<td>18G needles</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>1&#189; inch needle, 1/syringe</td>
</tr>
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<td>Dissection scissors</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>4" sharp straight blades</td>
</tr>
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<td>Fertilized Eggs</td>
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windowing chicken eggs for developmental studies

開発の研究が大幅に実験モデルとしてニワトリ胚の使用によって促進されています。胚のアクセシビリティの容易さは、ニワトリウズラのキメラとフォーカル色素のラベリングを含めて、いくつかのアプローチを、使用して細胞の運命をマッピングするための実験を​​可能にした。さらに、それはタンパク質でコーティングされたビーズとOVOエレクトロで使用してプラスミドDNAの導入の配置などのいくつかの種類の分子摂動、することができます。これらの実験は、中枢および末梢神経系の発達に重要なデータが得られている。これらの研究の多くの場合、それは卵の殻を開いて、胚の成長を乱すことなく、それをrecloseする必要があります。胚は、卵殻を再度開くことにより、連続した発達段階で調べることができます。鶏の卵を開くためのいくつかの優れた方法がありますが、この記事では、長い生存期間のために最適化されている一つの方法を示しています。この方法では、卵はその側にかかっていると、小さなウィンドウがシェルにカットされます。実験手順の後に、シェルはその開発期間中の卵をカバーするために使用されます。卵殻を覆う透明なプラスチックテープは、胚を保護し、潜伏期間の残りの中に水和を保持するのに役立ちます。このメソッドは、インキュベーションの2日間の始まりを使用されており、成熟した胚古くから生存を可能にした。

Watch this Scientific Journal Video about Windowing Chicken Eggs for Developmental Studies at JoVE.com

Windowing Chicken Eggs for Developmental Studies

胚のアクセシビリティの容易さは、ニワトリウズラのキメラとフォーカル色素のラベリングを含めて、いくつかのアプローチを、使用して細胞の運命をマッピングするための実験を​​可能にした。この記事では、長い生存時間に対して最適化されている1個の卵の調製方法を示しています。

発達研究のためのウィンドウ鶏卵

The study of development has been greatly aided by the use of the chick embryo as an experimental model.  The ease of accessibility of the embryo has ...

windowing-chicken-eggs-for-developmental-studies

Research

JoVE Journal

Biology

Obtaining Eggs from Xenopus laevis Females

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

アフリカツメガエル女性から卵子を入手

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, ...

生物学

Xenotransplantation of Human Stem Cells into the Chicken Embryo

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the behavior of cells in a standardized in vivo environment. The main advantages of the chicken embryo include low cost, high accessibility, ease of surgical manipulation and lack of a fully developed immune system. Xenotransplantation into chicken embryos can provide valuable information about cell proliferation, differentiation and behavior, the responses of cells to signals in defined embryonic tissue niches, and tumorigenic potential. Transplanting cells into chicken embryos can also be a step towards transplantation experiments in other animal models. Recently the chicken embryo has been used to evaluate the neurogenic potential of human stem and progenitor cells following implantation into neural anlage1-6. In this video we document the entire procedure for transplanting human stem cells into the developing central nervous system of the chicken embryo. The procedure starts with incubation of fertilized eggs until embryos of the desired age have developed. The eggshell is then opened, and the embryo contrasted by injecting dye between the embryo and the yolk. Small lesions are made in the neural tube using microsurgery, creating a regenerative site for cell deposition that promotes subsequent integration into the host tissue. We demonstrate injections of human stem cells into such lesions made in the part of the neural tube that forms the hindbrain and the spinal cord, and into the lumen of the part of the neural tube that forms the brain. Systemic injections into extraembryonic veins and arteries are also demonstrated as an alternative way to deliver cells to vascularized tissues including the central nervous system. Finally we show how to remove the embryo from the egg after several days of further development and how to dissect the spinal cord free for subsequent physiological, histological or biochemical analyses.

In this paper we present a method for transplanting human stem cells into various regions of the central nervous system of the chicken embryo. This provides an in vivo model for assessing the proliferation and differentiation of various types of human stem cells in embryonic tissue environments.

ニワトリ胚へのヒト幹細胞の異種移植

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

異種哺乳動物細胞で発現したイオンチャネルの変異導入と機能解析

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

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.

して、古いニワトリ胚への遺伝子導入<em&gt; EX OVO</em&gt;エレクトロポレーション

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

Separation of Spermatogenic Cell Types Using STA-PUT Velocity Sedimentation

Mammalian spermatogenesis is a complex differentiation process that occurs in several stages in the seminiferous tubules of the testes. Currently, there is no reliable cell culture system allowing for spermatogenic differentiation in vitro, and most biological studies of spermatogenic cells require tissue harvest from animal models like the mouse and rat. Because the testis contains numerous cell types - both non-spermatogenic (Leydig, Sertoli, myeloid, and epithelial cells) and spermatogenic (spermatogonia, spermatocytes, round spermatids, condensing spermatids and spermatozoa) - studies of the biological mechanisms involved in spermatogenesis require the isolation and enrichment of these different cell types. The STA-PUT method allows for the separation of a heterogeneous population of cells - in this case, from the testes - through a linear BSA gradient. Individual cell types sediment with different sedimentation velocity according to cell size, and fractions enriched for different cell types can be collected and utilized in further analyses. While the STA-PUT method does not result in highly pure fractions of cell types, e.g. as can be obtained with certain cell sorting methods, it does provide a much higher yield of total cells in each fraction (~1 x 108 cells/spermatogenic cell type from a starting population of 7-8 x 108 cells). This high yield method requires only specialized glassware and can be performed in any cold room or large refrigerator, making it an ideal method for labs that have limited access to specialized equipment like a fluorescence activated cell sorter (FACS) or elutriator.

The STA-PUT method allows for the separation of different populations of spermatogenic cells based on size and density.

STA-PUTの速度沈降を使用して造精細胞型の分離

Mammalian spermatogenesis is a complex  differentiation process that occurs in several stages in the seminiferous  tubules of the testes. Currently, there ...

A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins within complex biological samples. Since then, western blotting methodology has become a common component of the molecular biologists experimental repertoire. A cursory search of PubMed using the term &#8220;western blot&#8221; suggests that in excess of two hundred and twenty thousand published manuscripts have made use of this technique by the year 2014. Importantly, the last ten years have seen technical imaging advances coupled with the development of sensitive fluorescent labels which have improved sensitivity and yielded even greater ranges of linear detection. The result is a now truly Quantifiable Fluorescence based Western Blot (QFWB) that allows biologists to carry out comparative expression analysis with greater sensitivity and accuracy than ever before. Many &#8220;optimized&#8221; western blotting methodologies exist and are utilized in different laboratories. These often prove difficult to implement due to the requirement of subtle but undocumented procedural amendments. This protocol provides a comprehensive description of an established and robust QFWB method, complete with troubleshooting strategies.

The advancement of western blotting using fluorescence has allowed detection of subtle changes in protein expression enabling quantitative analyses. Here we describe a robust methodology for detection of a range of proteins across a variety of species and tissue types. A strategy to overcome common technical problems is also provided.

トラブルシューティングの方法と現代の定量的蛍光ウェスタンブロッティングへのガイド

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins ...

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here protocols are presented for preparing embryos and adult C. elegans animals for short- and long-term time-lapse microscopy and methods for tracking and quantification of developmental processes. The methods presented are all based on C. elegans strains available from the Caenorhabditis Genetics Center and on open-source software that can be easily implemented in any laboratory independently of the microscopy system used. A reconstruction of a 3D cell-shape model using the modelling software IMOD, manual tracking of fluorescently-labeled subcellular structures using the multi-purpose image analysis program Endrov, and an analysis of cortical contractile flow using PIVlab (Time-Resolved Digital Particle Image Velocimetry Tool for MATLAB) are shown. It is discussed how these methods can also be deployed to quantitatively capture other developmental processes in different models, e.g., cell tracking and lineage tracing, tracking of vesicle flow.

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Here it is shown how to track and quantify developmental processes in C. elegans. The methods presented are based on open-source tools that can be easily implemented. It is demonstrated how to reconstruct 3D cell-shape models, how to manually track subcellular structures, and how to analyze cortical contractile flow.

の発達過程を追跡し、定量化<em&gt; C。エレガンス</em&gt;オープンソースのツールを使用しました

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here ...

Laser-assisted Lentiviral Gene Delivery to Mouse Fertilized Eggs

Lentiviruses are efficient vectors for gene delivery to mammalian cells. Following transduction, the lentiviral genome is stably incorporated into the host chromosome and is passed on to progeny. Thus, they are ideal vectors for creation of stable cell lines, in vivo delivery of indicators, and transduction of single cell fertilized eggs to create transgenic animals. However, mouse fertilized eggs and early stage embryos are protected by the zona pellucida, a glycoprotein matrix that forms a barrier against lentiviral gene delivery. Lentiviruses are too large to penetrate the zona and are typically delivered by microinjection of viral particles into the perivitelline cavity, the space between the zona and the embryonic cells. The requirement for highly skilled technologists and specialized equipment has minimized the use of lentiviruses for gene delivery to mouse embryos. This article describes a protocol for permeabilizing the mouse fertilized eggs by perforating the zona with a laser. Laser-perforation does not result in any damage to embryos and allows lentiviruses to gain access to embryonic cells for gene delivery. Transduced embryos can develop into blastocyst in vitro, and if implanted in pseudopregnant mice, develop into transgenic pups. The laser used in this protocol is effective and easy to use. Genes delivered by lentiviruses stably incorporate into mouse embryonic cells and are germline transmittable. This is an alternative method for creation of transgenic mice that requires no micromanipulation and microinjection of fertilized eggs.

Mouse fertilized eggs and early stage embryos are protected by the zona pellucida, a glycoprotein matrix that forms a barrier against gene delivery. This article describes a protocol for perforating the zona with a laser to transduce embryonic cells with lentiviral vectors and to create transgenic mice.

マウスへのレーザ レンチ ウイルス遺伝子導入受精卵

Lentiviruses are efficient vectors for gene delivery to mammalian cells. Following transduction, the lentiviral genome is stably incorporated into the ...

Measuring Photophysiology of Attached Stage of Colacium sp. by a Cuvette-Type Fast Repetition Rate Fluorometer

Fast repetition rate fluorometer (FRRf) is a beneficial method for measuring photosystem II (PSII) photophysiology and primary productivity. Although FRRf can measure PSII absorption cross-section (&#963;PSII), maximum photochemical efficiency (Fv/Fm), effective photochemical efficiency (Fq&#8242;/Fm&#8242;), and non-photochemical quenching (NPQNSV) for various eukaryotic algae and cyanobacteria, almost all FRRf studies to date have focused on phytoplankton. Here, the protocol describes how to measure PSII photophysiology of an epizoic alga Colacium sp.&#160;Ehrenberg 1834 (Euglenophyta), in its attached stage (attached to zooplankton), using cuvette-type FRRf. First, we estimated the effects of substrate zooplankton (Scapholeberis mucronata O.F. M&#252;ller 1776, Cladocera, Daphniidae) on baseline fluorescence and &#963;PSII, Fv/Fm, Fq&#8242;/Fm&#8242;, and NPQNSV of planktonic Colacium sp. To validate this methodology, we recorded photophysiology measurements of attached Colacium sp. on S. mucronata and compared these results with its planktonic stage. Representative results showed how the protocol could determine the effects of calcium (Ca) and manganese (Mn) on Colacium sp. photophysiology and identify the various effects of Mn enrichment between attached and planktonic stages. Finally, we discuss the adaptability of this protocol to other periphytic algae.

Measuring Photophysiology of Attached Stage of Colacium sp. by a Cuvette-Type Fast Repetition Rate Fluorometer

Fast repetition rate fluorometer (FRRf) is a beneficial method for measuring photosystem II photophysiology and primary productivity. Here we describe a protocol to measure PSII photophysiology of epizoic alga, Colacium sp. on substrate zooplankton using cuvette-type FRRf.

キュベット型高速繰り返し速度蛍光光度計による コラシウム 属の付着ステージの光生理機能の測定

Fast repetition rate fluorometer (FRRf) is a beneficial method for measuring photosystem II (PSII) photophysiology and primary productivity. Although FRRf ...

ニワトリレンズ開発におけるタンパク質発現の研究

Microinjection of Recombinant RCAS(A) Retrovirus into Embryonic Chicken Lens

Embryonic chicken (Gallus domesticus) is a well-established animal model for the study of lens development and physiology, given its high degree of similarity with the human lens. RCAS(A) is a replication-competent chicken retrovirus that infects dividing cells, which serves as a powerful tool to study the in situ expression and function of wild-type and mutant proteins during lens development by microinjection into the empty lumen of lens vesicle at early developmental stages, restricting its action to surrounding proliferating lens cells. Compared to other approaches, such as transgenic models and ex vivo cultures, the use of an RCAS(A) replication-competent avian retrovirus provides a highly effective, rapid, and customizable system to express exogenous proteins in chick embryos. Specifically, targeted gene transfer can be confined to proliferative lens fiber cells without the need for tissue-specific promoters. In this article, we will briefly overview the steps needed for recombinant retrovirus RCAS(A) preparation, provide a detailed, comprehensive overview of the microinjection procedure, and provide sample results of the technique.

著者スポットライト:ニワトリ胚水晶体におけるRCAS(A)レトロウイルスのマイクロインジェクションによる水晶体の発生と機能の調査 

This protocol paper describes the methodology of embryonic chicken lens microinjection of an RCAS(A) retrovirus as a tool for studying in situ function and expression of proteins during lens development.

組換えRCAS(A)レトロウイルスのニワトリ胚水晶体へのマイクロインジェクション

Embryonic chicken (Gallus domesticus) is a well-established animal model for the study of lens development and physiology, given its high degree of ...