에그 처리 <ol><li> 계란이 생존에 상당한 손실 (작은 와인 쿨러는 이러한 용도에 이상적입니다)없이 부화하기 전에 최대 1 주일 13 ° C에 보관 수 있습니다. </li><li> 이전 부화하기 위해 달걀이 인큐베이터는 37.8로 설정 humidified 닭고기에 다음 상온, 장소에 따뜻한 있도록 ° C (100 ° F). </li><li> 우리는 일반적으로 배아가 햄버거와 해밀턴 단계 10에 도달 인큐베이션의 시작에서 태아에게 약 48 시간 electroporate. </li><li> 부화의 시간 원하는 햄버거와 해밀턴 단계에 따라 다를 수 있습니다. 온도가 최적 배양 온도에서 중요하고 편차 것은 배양 시간 및 감소 배아 생존을 변경합니다. </li><li> 부화 기간 동안, 계란은 배아가 제대로 electroporation에 대한 위치를되도록 자신의 양쪽에 보관해야합니다. 난자는 노른자 위에 떠되며 어디자를 알고 그래서 윈도우 전에 연필로 계란의 맨 위에 표시하기 위해 도움이됩니다. </li></ol> 준비 <ol><li> 37 ° C.에 따뜻한 Leibovitz L - 15 미디어 바늘에 유리 모세관을 당기세요. 위치 전극과 micromanipulator 및 펄스 발생기에 전극을 연결합니다. </li></ol> 윈도우 <ol><li> 피어스 계란의 작은 끝에 껍질, 큰 구멍 바늘로 주사를 사용합니다. </li><li> 노른자의 중단을 방지하기 위해 조심스럽게 아래 바늘 가리키며, 알부민의 5ml에 대한 제거합니다. </li><li> 테이프의 작은 조각으로 구멍을 밀봉합니다. </li><li> 테이프의 또 다른 조각 (4X4 cm 정도)와 계란의 상단 커버. </li><li> 곡선 가위를 사용하고 배아를 방해하지를 돌보는 작업에 창문을 제공하기 위해 충분 큰 구멍을 뚫고. </li></ol> 옵션 : 신경 튜브를 시각화하려면, 배 (胚) 아래에 26 게이지 바늘 (혈액 링 밖에서 배아 아래에 바늘을 삽입)와 잉크 솔루션을 주입. 인도 잉크는 행크스 또는 유사한 살균 버퍼 솔루션 1시 5분 희석해야합니다. 사출 및 Electroporation <ol><li> 핀셋을 사용하여 원하는 직경으로 모세관 팁을 브레이크 </li><li> 입 피펫을 사용하여 플라스미드 / 빠른 그린 염색과 모세를로드합니다. </li><li> 여러분쪽으로 머리와 배아를 위치와 얕은 각도로 신경 튜브에 바늘을 삽입합니다. </li><li> 염료가 전체 공간을 채우고까지 신경 튜브의 루멘에 플라스미드 솔루션을 주사. </li></ol> 참고 : 다음 끝 큰 신경 튜브 직경을하지 않도록주의하십시오. 일반적으로 최적의 팁 크기는 모세관에 액체를 뽑아 얼마나 쉽게 또는 하드에 의해 달성되었는지 말할 수있을 것입니다. 극단적인 저항이있다면, 시작은 아마도 너무 작습니다 그리고 끝까지 높은 망가해야합니다. 거의 또는 전혀 저항이있다면, 오프닝이 너무 큽니다 새로운 주사 바늘을 사용해야합니다. <ol><li> 배아에서 멸균 L - 15 미디어의 장소는 1-2 방울. </li><li> 당장 1 초 간격으로 50 mseconds 위해 10-24 볼트의 신경 튜브에 평행 배아의 양쪽에 전극, 및 펄스 5 번 놓으십시오. 그것이 제대로 작동하면 전극 근처에 거품을 볼 수 있습니다. </li><li> 조심스럽게, 전극을 제거 배아 위에 L - 15의 5 방울을 넣고 테이프로 계란을 밀봉. 건조가 electroporation 다음 배아 손실에 비만으로 계란이 잘, 밀봉되어 있는지 확인합니다. </li><li> 인큐베이터에 다시 플레이스 계란, 창 측면까지, 그들이 H &amp;​​ H 단계를 원하는 도달할 때까지 알을 품다. </li></ol>

<ol>
	<li>Itasaki, N., Bel-Vialar, S., Krumlauf, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&apos;Shocking&apos;+developments+in+chick+embryology:+electroporation+and+in+ovo+gene+expression.">&apos;Shocking&apos; developments in chick embryology: electroporation and in ovo gene expression.</a> Nat Cell Biol. 1, 203-207 (1999).</li></ol>

The authors have nothing to disclose.

이 프로토콜은 HH10 여자는 태아의 신경 튜브 electroporation 단계 가이드에 의해 단계를 제공합니다. 기술을 보여주는 이외에도, 스토리지 및 계란의 취급에 대한 지침도 제공됩니다. 이 기술은 유전자 분석을위한 어플 리케이션의 광범을 가지고 있으며, 실험의 용이성과 낮은 비용으로 많은 실험실 위해이 가능합니다.

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<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>Chicken egg incubator</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>humidified, set to 37.80&deg;C (100F). </td>
</tr>
<tr>
<td>Pulse generator </td>
<td>&nbsp;</td>
<td>Genetronics </td>
<td>BTX T820 </td>
<td>Square wave generator or comparable</td>
</tr>
<tr>
<td>Electrodes</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>our electrodes were made from 0.25 mm diameter platinum wire and were 5 mm long and spaced 1 mm apart</td>
</tr>
<tr>
<td>Connecter cables</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Micromanipulator</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Dissecting scope </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>with large working distance</td>
</tr>
<tr>
<td>Glass capillaries </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>and capillary puller or commercial glass needles</td>
</tr>
<tr>
<td>Leibovitz&#x2019;s L-15 media</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>chicken eggs</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>fertilized</td>
</tr>
<tr>
<td>Curved scissors</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>10 ml syringe</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Large bore needle </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>16-18G</td>
</tr>
<tr>
<td>Mouth pipette</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>India ink </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Insulin syringe/syringe </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Plasmid DNA </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&#x2265; 1&#181;g/&#181;l in sterile TE or 1X PBS containing 0.05% Fast Green dye</td>
</tr>		</tbody>
		</table>
		</div>
			<div>
					</div>

in ovo electroporations of hh stage 10 chicken embryos

대형 크기와 닭 배아의 외부 개발 오래 발달 생물학 연구에 가치있는 도구가 만들었습니다. 분자 생물 학적 기법의 도래와 함께, 여자가 유전자 조절과 기능을 연구하는 유용한 시스템이되고있다. 개발 닭 배아에 DNA 또는 RNA 구조를 electroporating함으로써, 유전자는 명시적 또는 생체내 유전자 기능의 분석을 위해 무너뜨렸다 수 있습니다. 마찬가지로 기자의 구성은 운명 매핑에 사용할 수 또는 putative 유전자 규제 요소를 검사합니다. 마우스에서 유사한 실험에 비해, 여자의 electroporation은 빠르고 쉽고 저렴한되는 장점이 있습니다. 이 비디오는 배아를 조작하기 위해 달걀 껍질에 창문을 만드는 방법 첫째 보여줍니다. 다음, 난자는 배아 아래 주입 인도 잉크의 희석 용액과 시각이다. 유리 바늘과 피펫은 개​​발 신경 튜브 다음, 백금 전극이 배아로 병렬 배치하고 짧은 전기 펄스가 펄스 발생기와 함께 관리되는에 DNA와 빠른 그린 염료를 주입하는 데 사용됩니다. 마지막으로 계란은 테이프로 밀봉하고 발전을위한 인큐베이터에 다시 배치. 또한, 비디오 적절한 계란 보관 및 처리를 보여줍 배아 손실 다음의 electroporation의 가능한 원인에 대해 설명합니다.

Watch this Scientific Journal Video about In Ovo Electroporations of HH Stage 10 Chicken Embryos at JoVE.com

In Ovo Electroporations of HH Stage 10 Chicken Embryos

비켜 electroporation의 여자는 조류 배아의 유전자 조작을 허용하는 기법입니다. 이 기법의 일반적인 응용 프로그램은 유전자와 putative 향상제 요소의 기능 분석을 포함합니다. 이 동영상은 HH 10 여자는 배아에서 신경 튜브 electroporation을 보여줍니다. 사출 기술과 적절한 계란 처리는 설명합니다.

HH 스테이지 10 치킨 태아의 비켜 Electroporations에서

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 ...

in-ovo-electroporations-of-hh-stage-10-chicken-embryos

Research

JoVE Journal

Biology

18.2K 조회수.  University of Chicago. 비켜 electroporation의 여자는 조류 배아의 유전자 조작을 허용하는 기법입니다. 이 기법의 일반적인 응용 프로그램은 유전자와 putative 향상제 요소의 기능 분석을 포함합니다. 이 동영상은 HH 10 여자는 배아에서 신경 튜브 electroporation을 보여줍니다. 사출 기술과 적절한 계란 처리는 설명합니다.

비디오: In Ovo Electroporations of HH Stage 10 Chicken Embryos

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.

Midgastrula 스테이지 Drosophila의 태아에서 문화의 연결 준비

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

연구

생물학

Windowing Chicken Eggs for Developmental Studies

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 allowed for experiments to map cell fates using several approaches, including chick quail chimeras and focal dye labeling.  In addition, it allows for molecular perturbations of several types, including placement of protein-coated beads and introduction of plasmid DNA using in ovo electroporation.  These experiments have yielded important data on the development of the central and peripheral nervous systems.  For many of these studies, it is necessary to open the eggshell and reclose it without perturbing the embryo's growth.  The embryo can be examined at successive developmental stages by re-opening the eggshell.  While there are several excellent methods for opening chicken eggs, in this article we demonstrate one method that has been optimized for long survival times.  In this method, the egg rests on its side and a small window is cut in the shell.  After the experimental procedure, the shell is used to cover the egg for the duration of its development.  Clear plastic tape overlying the eggshell protects the embryo and helps retain hydration during the remainder of the incubation period.  This method has been used beginning at two days of incubation and has allowed survival through mature embryonic ages.

The ease of accessibility of the embryo has allowed for experiments to map cell fates using several approaches, including chick quail chimeras and focal dye labeling. In this article we demonstrate one egg preparation method that has been optimized for long survival times.

발달 연구를위한 윈도우 치킨 계란

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 ...

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.

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.

초기 치킨 태아의 성장 팩터 - 코팅 비즈 배치

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

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.

병아리 예 비켜 문화와 예 비켜 CAM 어세이 : 그게 정말로 작동하는 방법

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.

의 전자 현미경을위한 배아의 준비 Drosophila 태아의 심장 관

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

In-vivo Centrifugation of Drosophila Embryos

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in buoyant density. However, when cells are disrupted prior to centrifugation, proteins and organelles in this non-native environment often inappropriately stick to each other. Here we describe a method to separate organelles by density in intact, living Drosophila embryos. Early embryos before cellularization are harvested from population cages, and their outer egg shells are removed by treatment with 50% bleach. Embryos are then transferred to a small agar plate and inserted, posterior end first, into small vertical holes in the agar. The plates containing embedded embryos are centrifuged for 30 min at 3000g. The agar supports the embryos and keeps them in a defined orientation. Afterwards, the embryos are dug out of the agar with a blunt needle. Centrifugation separates major organelles into distinct layers, a stratification easily visible by bright-field microscopy. A number of fluorescent markers are available to confirm successful stratification in living embryos. Proteins associated with certain organelles will be enriched in a particular layer, demonstrating colocalization. Individual layers can be recovered for biochemical analysis or transplantation into donor eggs. This technique is applicable for organelle separation in other large cells, including the eggs and oocytes of diverse species.

In-vivo Centrifugation of Drosophila Embryos

We describe a method to separate organelles by density in living Drosophila embryos. Embryos are embedded in agar and centrifuged. This technique yields reproducible separation of major organelles along the anterior-posterior embryo axis. This method facilitates colocalization experiments and yields organelle fractions for biochemical analysis and transplantation experiments.

Drosophila의 배아들 중에서 - 생체내의 원심 분리

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in ...

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for discovering new therapeutic strategies. Advancements in imaging technology have enabled quantitative investigation of the organization and maturing of the body plan, but later stage embryonic morphogenesis is less clear. Chicken embryos are an attractive vertebrate animal model system for this application because of its ease of culture and surgical manipulation. Early embryos can be cultured for a short time on filter paper rings, which enables complete optical access for cell patterning and fate studies1,2. Studying advanced developmental processes such as cardiac morphogenesis are traditionally performed through a window of the eggshell3-5, but this technique limits optical access due to window size. We previously developed a simple method to culture whole embryos ex-ovo on hexagonal weigh boats for up to 10 days, which enabled high resolution imaging via ultrasonography6,7. These cultures were difficult to transport, limiting the types of imaging tools available for live experiments. We here present an improved shell-less culture system with a cost-effective, portable environmental chamber. Eggs were cracked onto a hammock created by a polyurethane membrane (cling wrap) affixed circumferentially to a plastic cup partially filled with sterile water. The dimensions of the circumference and depth of the hammock were both critical to maintain surface tension, while the mechanics of the hammock and water beneath helped dampen vibrations induced by transportation. A small footprint circulating water bath was also developed to enable continuous temperature control during experimentation. We demonstrate the ability to culture embryos in this way for at least 14 days without morphogenic defect or delay and employ this system in several microsurgical and imaging applications.

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

In this article, we present a simple methodology to enable long-term ex-ovo avian embryo culture. This technique is ideal for longitudinal experimentation requiring complete optical accessibility and/or sterile transportation in avian embryos.

전 비켜</em이미징 및 미세 애플 리케이션에 적합> 치킨의 배아 문화 시스템

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for ...

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

생활의 이미징 셀 모양 변경 Drosophila 태아

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

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.

초기 마우스 배아를 사용하는 조직 특정 유전자에 대한 염색질의 Immunoprecipitation 분석

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

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.

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.

배아 칙의 망막에서 비켜 Electroporation에

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