우리는 자신의 촬영에서 작업이 원고의 비디오 부분을 편집 마운트 세인트 빈센트 대학의 폴 포이 리어, 미디어 프로듀서, 감사의 말씀을 전합니다. 우리는 자금에 대한 캐나다의 자연 과학 및 공학 연구위원회를 인정합니다.

참고 : 모든 소모품은 표 1에 나열되어 있습니다. 1. 닭 배아를 저장 <ol><li> 약 40 %의 습도 루스가 37 ℃로 수평 루스 균주의 닭 알을 품어 한 번 또는 두 번 매일 계란을 켭니다. 달걀을 돌리면 껍질에 부착 배아를 방지하는 것이 중요하다. </li><li> 4 O C에서 계란을 유지, 이전에, 그렇지 않으면 배아는 노른자 질량에 복부를 위치 할 것이며, 또한 3 단계에서 계란을 여는 손상되는 바와 같이 문화를 설정하여 24 시간에 계란을 설정하지 마십시오 &quot;정지&quot;개발에 배양하기 전에 이하 1 주일도하지만이 적합하지 않습니다. </li></ol> 2. 닭 배아를 준비 <ol><li> 햄버거와 해밀턴 (12) 스테이징 테이블을 사용하여 닭 배아를 스테이지. 전 비켜 배양를 설정하는 이상적인 단계는 HH 단계 19 ~ 20 (배양 3 약 일) 벡입니다ause이는 머리가 53 HPF에서 전환 직후입니다. 주 : HH 단계 19-20는 다음의 형태 학적 특성을 특징으로한다 : somites 꼬리의 과반수로 연장되어 있지만, 꼬리의 끝은 꼬리 봉오리가 말려, 비분 남아 allantois이 작고 맥관 한계가 다리 싹이 날개 - 싹이보다 큰하고 눈은 착색 또는 회색 빛 색조를 가지고있다. </li></ol> 3. 쉘에서 배아를 제거 <ol><li> 쉘에서 배아를 제거하기 전에, 70 % 에탄올로 쉘을 스프레이하고 건조 할 수 있습니다. 이 배아를 손상되므로 신속하게 계란을 끄지 마십시오. </li><li> 조심하는 것은 조심스럽게 아래쪽에 계란을 균열, 달걀의 방향을 변경하지 (즉, 배양 중에 복부이었다 측)과 멸균에 배아를 해제 보트 (88 X 88 X 23mm)에 무게. 닦아 70 % 에탄올 보트의 무게. 노른자 자루가 손상되지 않았는지 확인하기 위해 배아를 검사합니다. 노른자 시간의 경우깨진로 생존하지 않으므로, 배아를 폐기합니다. </li><li> 그것이 가능한 지 확인하기 위해 배아를 관찰; 심장 박동, 혈관은 정상 나타나고, 명백한 이상이 존재하지 않습니다. 무게 보트의 가장자리가 건조되었는지 확인합니다. </li><li> 피펫을 사용하여 감염을 방지하기 위해 알부민의 상단에 페니실린 / 스트렙토 마이신의 40 μL (5,000 단위의 페니실린, 1 mL 당 5 mg을 스트렙토 마이신)를 놓습니다. </li></ol> 4. 습도 챔버 준비 <ol><li> 킴 와이프 ​​- 및 / 또는 멸균 플라스틱 용기 (12 X 12 X 6cm)의 바닥면으로 이루어지는 흡수 패드의 작은 스택을 놓고 70 % 에탄올로 닦았다. </li><li> 김 와이프 ​​및 / 또는면 (물 약 150 ml)에 축축하게 멸균 증류수를 추가합니다. </li></ol> 5. 예 비켜 문화 조립 <ol><li> 축축한 패딩의 상단에있는 배아를 포함하는 무게 보트를 놓습니다. 그런 다음, t의 상단에 사각형 멸균 페트리 접시 (9.5 X 9.5 cm)의 장소 반그는 느슨한 뚜껑을 형성하기 위해 보트의 무게. </li><li> 그 뚜껑이있는 플라스틱 용기를 커버. 여전히 챔버 내부에 좋은 공기 흐름을 허용하는 부분 도장을 보장, 뚜껑의 두 모서리를 누르십시오. </li><li> 조심스럽게 원하는 단계까지 37 O를 C 배양기에 설치를 놓습니다. </li><li> 제어 습도 배양기를 사용할 수없는 경우, 습도를 제어 할 수 있도록하기 위해 인큐베이터에서 물 용기를 놓습니다. 습도 챔버 및 배양기에서 모든 물을 소독 잠복기 동안 필요에 따라 보충. 40 %의 습도가 이상적입니다. </li></ol>

<ol>
	<li>Auerbach, R., Kubai, L., Knighton, D., Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+procedure+for+the+long-term+cultivation+of+chicken+embryos.">A simple procedure for the long-term cultivation of chicken embryos.</a> Dev. Biol. 41, 391-394 (1974).</li><li>Gennaro, L. D., Packard, D. S., Stach, R. W., Wagner, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+differentiation+of+chicken+embryos+in+simplified+shell-less+cultures+under+ordinary+conditions+of+incubation.">Growth and differentiation of chicken embryos in simplified shell-less cultures under ordinary conditions of incubation.</a> Growth. 44, 343-354 (1980).</li><li>Tufan, C. A., Akdogan, I., Adiguzel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shell-less+culture+of+the+chick+embryo+as+a+model+system+in+the+study+of+developmental+neurobiology.">Shell-less culture of the chick embryo as a model system in the study of developmental neurobiology.</a> Neuroanat. 3, 8-11 (2004).</li><li>Duench, K., Franz-Odendaal, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BMP+and+Hedgehog+signaling+during+the+development+of+scleral+ossicles.">BMP and Hedgehog signaling during the development of scleral ossicles.</a> Dev. Biol. 365 (1), 251-258 (2012).</li><li>Datar, S., Bhonde, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shell-less+Chick+Embryo+Culture+as+an+Alternative+in+vitro+Model+to+Investigate+Glucose-Induced+Malformation+in+Mammalian+Embryos.">Shell-less Chick Embryo Culture as an Alternative in vitro Model to Investigate Glucose-Induced Malformation in Mammalian Embryos.</a> Rev Diabet Stud. 2 (4), 221-227 (2005).</li><li>Fisher, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chick+embryos+in+shell-less+culture.+Tested+studies+for+laboratory+teaching.">Chick embryos in shell-less culture. Tested studies for laboratory teaching.</a> , (1983).</li><li>Dunn, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technique+for+shell-less+culture+of+the+72-hour+avian+embryo).">Technique for shell-less culture of the 72-hour avian embryo).</a> Poultry Science. 53, 409-412 (1974).</li><li>Yalcin, H., Shekhar, A., Rane, A. A., Butcher, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ex-ovo+chicken+embryo+culture+system+suitable+for+imaging+and+microsurgery+applications.">An ex-ovo chicken embryo culture system suitable for imaging and microsurgery applications.</a> J. Vis. Exp. (44), (2010).</li><li>Silver, P. H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Special+problems+of+experimenting+in+ovo+on+the+early+chick+embryo,+and+a+solution.">Special problems of experimenting in ovo on the early chick embryo, and a solution.</a> J Embryol Exp Morph. 8 (4), 369-375 (1960).</li><li>Spurlin, J., Lwigale, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+technique+to+increase+accessibility+to+late-stage+chick+embryos+for+in+ovo+manipulations.">A technique to increase accessibility to late-stage chick embryos for in ovo manipulations.</a> Dev. Dyn. 242 (2), 148-154 (2012).</li><li>Dorrell, M. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+ovo+model+for+directly+visualizing+chicken+embryo+development.">Ex ovo model for directly visualizing chicken embryo development.</a> American Biology Teacher. 74 (9), (2012).</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.">A series of normal stages in the development of the chick embryo.</a> J. Morph. 88, 49-92 (1951).</li><li>Drossopoulou, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+for+anteroposterior+patterning+of+the+vertebrate+limb+based+on+sequential+long-and+short-range+Shh+signaling+and+Bmp+signaling.">A model for anteroposterior patterning of the vertebrate limb based on sequential long-and short-range Shh signaling and Bmp signaling.</a> Development. , 127-1337 (2000).</li><li>Sys, G. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+in+ovo+CAM-assay+as+a+xenograft+model+for+sarcoma.">The in ovo CAM-assay as a xenograft model for sarcoma.</a> J. Vis. Exp. (77), e50522 (2013).</li><li>Franz-Odendaal, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+understanding+the+development+of+scleral+ossicles+in+chicken,+Gallus+gallus.">Towards understanding the development of scleral ossicles in chicken, Gallus gallus.</a> Dev. Dyn. 237, 3240-3251 (2008).</li></ol>

저자는이 논문에서 제시된 정보에 관해서 더 경쟁 금융 이익이 없습니다.

배양을 모두 윈도 비켜 예 장점과 도전이있다. 여기에서 우리는 장점과 방법 비켜 스티로폼 컵 전 아래에 표시된 방법 비켜 우리의 최적화 된 예에 윈도우 방법의 문제를 비교합니다. 우리의 방법은 늦게 개발의 단계 및 방법 1, 2 비켜 전통적인 예에 대한 우리의 개선에 조작과 병아리 배아를 관찰 할 수 있도록, 3 학부 교육 실험실 클래스에서 사용하는 것이 또한 매우 간단합니다. 이 수행 할 간단하고 개발의 후반 단계 (HH 41 ~ 42)에 우수한 생존 성을 가지고 있기 때문에 많은 연구자들이 닭 개발을 연구하기 위해 윈도우 방법을 선호하지만, 그것은 한계가있다. 그 중 가장 큰 무대 후 HH (30) HH 단계 (30) 후 전체 배아를 볼 수있는 제한된 능력이다, 달걀 껍질에있는 창은 성장, 이동 배아를 이용하기 위해서는 매우 큰 필요가있다. 이 대형 윈도우는 DIFF입니다icult 완전히 (불임과 생존의 문제로 이어지는), 도전 할 수있는 계란 내부에 좋은 조명을 얻기 밀봉합니다. 또한, 고급 단계의 배아는 창을 통해 크고 어려운 액세스 (또는 조작)입니다. 여기에 설명 된 방법 비켜 전 배아에 대한 모든 자유로운 액세스를 제공합니다. 계량 보트 습도 챔버에 배치되면, 전체 셋업은 매우 안정하다. 전체 습도 챔버 해부 현미경 아래에 배치 될 수 있고, 고배율은 전체 배아를 관찰하는데 사용될 수있다. 이에 비해, 스티로폼 컵 방법 높이 습도 챔버 (컵 높이)를 가지며, 이것은 매우 어려운 목표 현미경 하에서 배치 할 수있다. 컵 디자인은 보트의 무게도 훨씬 덜 안정된 평면보다 이러한 컵은 쉽게 뒤집히는 할 수 있습니다. 방법 비켜 예에 대한 우리의 개선은 교실이나 실험실 환경에서 사용하는이 방법은 간단하고 보안 목표 명세서를 할 수 있습니다HH (40)에 이르기까지 단계 HH 19 배아에 대한 모든 권한을 가지고 udent; 주요 기관 형성이 발생하면 스테이지. 방법 비켜 우리의 전직의 가장 큰 단점은 불임이다. 감염을 예방하기 위해, 우리의 방법은 항생제의 첨가를 포함한다. 항생제의 투여 량은 이후 또 다른 단계에서 배아를들 수있다. 배양 셋업 동안 투여 항생제는 배아 발달에 영향을 미치는 것으로 보이지 않는다. 극적으로 생존 성을 높일 수 있습니다 사용하기 전에 70 % 에탄올로 용기를 살균. 전반적으로, 방법 비켜 전 개발의 접근이나 무대에 제한없이 배아를보고하고 조작하기에 이상적입니다. 이전 연구는 배아에게 전 비켜을 배양하는 것은 부정적인 4,15을 패턴에 영향을주지 않는 것으로 나타났습니다. 예를 들어, 우리는 달걀 껍질의 부재에도 불​​구하고 패터닝 또는 골격의 유도에 어떤 변화를 볼 수 없습니다. 우리의 개선 방법은 여러 C를 해결방법 1, 2, 3 인 오보 전류 전통적인 EX와 병행 hallenges.

Ex ovo culturing has played an important role in the study of development of the chicken1, 2. This culturing method has been used to study neurological diseases, limb development, craniofacial development, and as a model to investigate malformations associated with diabetes 3, 4, 5.
There are many variations to the ex ovo technique. The most common approach is to use a Styrofoam cup6,7,8 or a glass bowl5. In these methods, the cup or bowl is lined with plastic wrap to cradle the embryo, a lid is placed on the cup, and the embryo is then placed in an incubator with appropriate humidity6. This set up however, can be technically challenging. The first challenge is the plastic wrap that is used to cradle the embryo. This wrap is difficult to work with and often does not adhere to the cup very well. To solve this problem, an elastic band is placed around the cup to hold the wrap in place. Despite this, the wrap can still slip, which is fatal to the embryo. The plastic wrap has the potential to tear or get punctured by forceps or needles that may be used during embryo manipulations and observations. Finally, this set-up is not very stable and students can easily knock the cups over. The height of the cups also makes it very difficult to place the embryo under a stereomicroscope, which has a limited objective to stage height. These challenges make it difficult for undergraduate students to work with live chick embryos in teaching labs, such as advanced developmental biology courses. 
The above challenges in the ex ovo method has meant that researchers turn to the windowing method 9,10 to view embryonic chick development. In this technique, a hole or &#8220;window&#8221; is made in the eggshell overlying the embryo. The hole can be re-sealed with tape or wax9 to allow for further embryonic development. Although the windowing method has some advantages, such as the ability to view embryonic development and easy maintenance, this method also has several limitations. The first is that the window needs to be fairly large in order to view the entire embryo (especially at late stages). Secondly, large windows are difficult to seal; an improper seal will lead to sterility and survivability problems. Using molten wax as a sealant adds another inconvenient and messy step to the protocol. Therefore, although the windowing method may be ideal for chick embryos at young stages (HH 11 &#8211; HH 27), viewing the entire embryo at late stages is not easily accomplished. 
Here, we describe an improved and simple ex ovo culturing technique11 that avoids the need for high tech equipment, is easy to handle under a stereomicroscope, gives the embryo enough support to perform microscopic manipulations, and enables researchers to view the growth of the embryo in its entirety well into the later stages of development (up to HH 40-41). With these advances in the ex ovo technique, individuals gain access to a more complete understanding of embryonic development. For instance, growth into later stages allows individuals to observe developmental processes that do not occur until this time point, such as ossification, feather development, and advanced limb and eye development. The entire embryo and extraembryonic membranes and vasculature are clearly visible. More advanced research can also be performed, such as, embryonic manipulations (i.e., implanting beadssoaked in inhibitors or inserting barriers between tissue layers), and researchers are then able to observe the effect of the manipulations in later stage embryos.

<table border="0" cellpadding="0" cellspacing="0" width="658">
	<tbody>
		<tr height="78">
			<td height="78" style="height:79px;width:177px;">Penicillin/Streptomycin</td>
			<td style="width:136px;">Sigma</td>
			<td style="width:152px;">P4458</td>
			<td style="width:192px;">Make small aliquots to avoid freeze/thaw events</td>
		</tr>
		<tr height="22">
			<td height="61" rowspan="3" style="height:61px;width:177px;">Square Petri Dish</td>
			<td rowspan="3" style="width:136px;">N/A</td>
			<td rowspan="3" style="width:152px;">N/A</td>
			<td rowspan="3" style="width:192px;">9.5 cm x 9.5 cm</td>
		</tr>
		<tr height="19">
		</tr>
		<tr height="20">
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:177px;">Weigh Boat</td>
			<td style="width:136px;">Fischer Scientific</td>
			<td align="right" style="width:152px;">8732113</td>
			<td style="width:192px;">88 x 88 x 23 mm</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:177px;">Ziplock container</td>
			<td style="width:136px;">Ziplock</td>
			<td style="width:152px;">N/A</td>
			<td style="width:192px;">12 cm x 12 cm x 6 cm</td>
		</tr>
	</tbody>
</table>

optimized ex-ovo culturing of chick embryos to advanced stages of development

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos model organisms, such as the chicken, are often used. The chicken is an excellent model organism due to its low cost and minimal maintenance, however they present observational challenges because they are enclosed in an opaque eggshell. In order to properly view the embryo as it develops, the shell must be windowed or removed. Both windowing and ex ovo techniques have been developed to assist researchers in the study of embryonic development. However, each of the methods has limitations and challenges. Here, we present a simple, optimized ex ovo culture technique for chicken embryos that enables the observation of embryonic development from stage HH 19 into late stages of development (HH 40), when many organs have developed. This technique is easy to adopt in both undergraduate classes and more advanced research laboratories where embryo manipulations are conducted.

방법 비켜이 전 늦게 개발의 단계 (HH 40 ~ 41) (그림 1A 및 1B)에 개발의 초기 단계 (HH 19/20)에서 배아의 관찰이 가능합니다. HH 19 ~ 20에서 문화를 설정하는 것은 문화의 배아의 생존을 증가시킨다. 생존의 문화와 단계 (21) 후 매우 낮은 (53 HPF 전) 선회 머리 이전에, 배아 적은 그대로 배아가 얻어진다 있도록 분리에 쉘에 더 집착하는 경향이있다. (약 40 % HH 40-41로 생존) 일반적으로, 문화 비켜 전에서 배아의 생존은 HH 35 ~ 36 (90~100%)에 높은이지만, 그것은 개발의 고급 단계에 드롭을한다. 그것은 그 발전을 보여왔다 있지만 또한 이러한 후반 단계에서 정상 나타나고 골격의 패터닝 4,15 영향을받지 않습니다, 우리는 사지 골화의 정도 또는 타이밍에 영향을 미칠 수있는 제안이 늦은 단계에서 구부러진 것을 관찰했다. 이다놀라운 일이 아니다 골화에 대한 쉘에서 칼슘의 필요성을 고려. 대조군과 비교 배아 발달의 상세한 비교가 진행 중이고,이 방법 종이의 범위를 벗어난다. 개발 전반에 걸쳐 배아에 대한 액세스를 얻는 것은 두 가지 이유에서 중요하다. 첫째로, 연구자들은 개봉 및 재 밀봉 창 않고도 매일 현상을 볼 수있다. 예를 들어, 개인은 여러 단계 (그림 1C 및 1D)에서 사지 눈의 발달을 관찰 할 수 있습니다. 둘째, 배양 비켜 예는, 태아에 이상 (무제한) 액세스 할 수 있도록함으로써 쉽게 조작 또는 수술을 가능하게한다. 배아 조작은 흔히, 실체 현미경을 사용하여 수행 될 필요가있다. 스티로폼 / 플라스틱 포장 방법 6,7,8 반대로 여기에 설명 배양 방법 비켜 예를 사용하여 설정을 쉽게 싶게 배치 된 배아어 현미경은 덜 깊은 전복되지 않습니다. 배아를 모호하게하는 어떠한 장벽 (난각)이 없기 때문에, 상기 챔버는 매우 안정하기 때문에이 필요하고 배아 액세스를 최적화하기를 회전하도록 정확하게 습도 챔버를 위치시키기 위해 연구를 가능하게한다. 전반적으로, 배아로부터받는 계량 보트 지지체는 약간 힘이 셋업은 축출없이 조작 중에 배아에 적용 할 수있다. 몇몇 발생 생물학 방법이 배양 계에서 배아에 대해 수행 될 수있다. 다음은 상세한 기관의 관찰 (예를 들면, 눈, 뇌 등) 또는 기형 에이전트의 조직 개발 (예를 들면, 혈관 성장) 나 응용 프로그램을 포함한다. 조작은 다른 조직 계층을 결합 또는 종양의 성장과 전이 (14) 공부의 효과를 연구하기 위해 융모 막 상 조직의 이식을 포함한다. 개발 과정에서 수행 인기있는 또 다른 조작은 BEA이다d 또는 장벽 주입. 비드 주입 연구원이 억제하거나 관심의 영역에서 로컬 유전자를 유도하기 위해 로컬 비드를 비드에 억제제 또는 요소를 흡수 한 후 이식 할 수 있습니다. 또이 문제는 아피 겔 또는 헤파린 비즈 (그림 2A)를 사용하여 수행됩니다. 예를 들어, 뼈 형태 형성 단백질 (BMP 형식)은 로컬 뼈 4 유래 신경 능선 (15)의 유도를 억제 할 수있는 동안, 또는 현상 상지에서 13 새싹. 비드 이식 후 배아 인큐베이터로 복귀 될 수 있으며, 이는 지속적으로 개발하는 것이다. 이 연구원은 경화성 반지의 공막, 이소골 형성의 억제 (그림 2B 및 2C) (15) (이 경우 BMP)에 특정 유전자의 억제의 하류 효과를 볼 수 있습니다. 유사하게, 장벽은 쉽게 조직 간 조직 시그널링 및 염료의 미세 주입법은 또한 용이하게 행할 수를 연구하기 위해 티슈 층 사이에 삽입 될 수있다. </p&gt; <img alt="그림 1" src="/files/ftp_upload/52129/52129fig1.jpg" /> 배아의 전 비켜보기 그림 1. (A) HH 단계 쉘에서 제거 후 20 병아리 배아, 혈관 배아 멤브레인 배아에 화살표가. (B) HH 단계 오픈 습도 챔버에서 40 배. (C) HH 단계 초기 사지 싹 (화살표)를 보여주는 35 배. (D) 등의 눈 (화살표)와 같은 구조의 나중 단계 개발을 보여주는 무대 HH 41 배. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52129/52129fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 2" src="/files/ftp_upload/52129/52129fig2.jpg" /> 소량의 그림 2. 예 주입 Experim 비드 배어ENT 4. (A) 배아 HH 스테이지 (35)는 비드 주입 하부 눈에 액세스하기 위해 위에 놓이는 멤브레인에서 찢어진 작은 구멍을 통해 노출. 결막 유두 인접 아피 겔 비드 고배율 인셋에 나타낸다. C의 스케일 바는 75 μm의입니다. (B) 알칼리 포스 파타 아제가 이소골의 반지의 격차를 보여주는 소량 비드 주입 (화살표), 이소골의 반지에 더 혼란을 보여주는 HH 38 (C) AP 스테인드 왼쪽 눈 (제어 후 오른쪽 눈 스테인드 )이 실험의 38 상세 Duench 프란츠 - Odendaal 4에서 제공되는 HH는. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52129/52129fig2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a>

Watch this Scientific Journal Video about Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development at JoVE.com

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Viewing and accessing the chicken embryo during development can be challenging. We have developed an ex ovo method that is simple, cost effective, and can easily be used in a classroom or research setting. This method provides access to the embryo into late stages of embryonic development (HH 40).

최적화<em&gt; 전 비켜</em&gt; 개발의 고급 단계에 병아리 배아의 배양

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos ...

optimized-ex-ovo-culturing-chick-embryos-to-advanced-stages

Nanyang Technological University

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JoVE Journal

Developmental Biology

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

13.5K 조회수.  Mount Saint Vincent University. Viewing and accessing the chicken embryo during development can be challenging. We have developed an ex ovo method that is simple, cost effective, and can easily be used in a classroom or research setting. This method provides access to the embryo into late stages of embryonic development (HH 40). 

비디오: Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying the biology of retinal neurons, particularly photoreceptors. The applications of this system go beyond basic research, as they can easily be adapted to high throughput technologies for drug development. However, genetic manipulation of retinal photoreceptors in these cultures has proven to be very challenging, posing an important limitation to the usefulness of the system. We have recently developed and validated an ex&#160;ovo plasmid electroporation technique that increases the rate of transfection of retinal cells in these cultures by five-fold compared to other currently available protocols1. In this method embryonic chick eyes are enucleated at stage 27, the RPE is removed, and the retinal cup is placed in a plasmid-containing solution and electroporated using easily constructed custom-made electrodes. The retinas are then dissociated and cultured using standard procedures. This technique can be applied to overexpression studies as well as to the downregulation of gene expression, for example via the use of plasmid-driven RNAi technology, commonly achieving transgene expression in 25% of the photoreceptor population. The video format of the present publication will make this technology easily accessible to researchers in the field, enabling the study of gene function in primary retinal cultures. We have also included detailed explanations of the critical steps of this procedure for a successful outcome and reproducibility.

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

Embryonic chick retinal cell cultures constitute valuable tools for the study of photoreceptor biology. We have developed an efficient gene transfer technique based on ex&#160;ovo plasmid electroporation of the retinas prior to culture. This technique considerably increases transfection efficiencies over currently available protocols, making genetic manipulation feasible for this system.

차 세포 배양 연구 병아리 망막에 효율적인 유전자 전송 :<em&gt; 전 비켜</em&gt; Electroporation에 접근

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying ...

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Forward Genetic Approach to Uncover Stress Resistance Genes in Mice &#8212; A High-throughput Screen in ES Cells

Phenotype-driven genetic screens in mice is a powerful technique to uncover gene functions, but are often hampered by extremely high costs, which severely limits its potential. We describe here the use of mouse embryonic stem (ES) cells as surrogate cells to screen for a phenotype of interest and subsequently introduce these cells into a host embryo to develop into a living mouse carrying the phenotype. This method provides (1) a cost effective, high-throughput platform for genetic screen in mammalian cells; (2) a rapid way to identify the mutated genes and verify causality; and (3) a short-cut to develop mouse mutants directly from these selected ES cells for whole animal studies. We demonstrated the use of paraquat (PQ) to select resistant mutants and identify mutations that confer oxidative stress resistance. Other stressors or cytotoxic compounds may also be used to screen for resistant mutants to uncover novel genetic determinants of a variety of cellular stress resistance.

Stress resistance is one of the hallmarks for longevity and is known to be genetically governed. Here, we developed an unbiased high-throughput method to screen for mutations that confer stress resistance in ES cells with which to develop mouse models for longevity studies.

앞으로 유전 접근은 마우스의 스트레스 저항성 유전자를 밝히기 위해서 - ES 세포에서 높은 처리량 화면

Phenotype-driven genetic screens in mice is a powerful technique to uncover gene functions, but are often hampered by extremely high costs, which severely ...

A Novel Ex Ovo Banding Technique to Alter Intracardiac Hemodynamics in an Embryonic Chicken System

The new model presented here can be used to understand the influence of hemodynamics on specific cardiac developmental processes, at the cellular and molecular level. To alter intracardiac hemodynamics, fertilized chicken eggs are incubated in a humidified chamber to obtain embryos of the desired stage (HH17). Once this developmental stage is achieved, the embryo is maintained ex ovo and hemodynamics in the embryonic heart are altered by partially constricting the outflow tract (OFT) with a surgical suture at the junction of the OFT and ventricle (OVJ). Control embryos are also cultured ex ovo but are not subjected to the surgical intervention. Banded and control embryos are then incubated in a humidified incubator for the desired period of time, after which 2D ultrasound is employed to analyze the change in blood flow velocity at the OVJ as a result of OFT banding. Once embryos are maintained ex ovo, it is important to ensure adequate hydration in the incubation chamber so as to prevent drying and eventually embryo death. Using this new banded model, it is now possible to perform analyses of changes in the expression of key players involved in valve development and to understand the role of hemodynamics on cellular responses in vivo, which could not be achieved previously.

A Novel Ex Ovo Banding Technique to Alter Intracardiac Hemodynamics in an Embryonic Chicken System

For the first time we present here a reproducible banding procedure to alter hemodynamics in the developing heart ex ovo. This is achieved by partially constricting the outflow tract (OFT).

소설<em&gt; 예 비켜</em&gt; 밴딩 기법 닭 배아 시스템에서 심장 내 혈류를 변경할

The new model presented here can be used to understand the influence of hemodynamics on specific cardiac developmental processes, at the cellular and ...

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of morphogenetic events, such as gastrulation2, neurulation3-5, somitogenesis6, heart bending7,8, and brain formation9-13, during early embryogenesis. Key to understanding morphogenetic processes is to follow them dynamically by time-lapse imaging. The acquisition of time-lapse movies of chick embryogenesis ex ovo has been limited either to short time windows or to the need for an incubator to control temperature and humidity around the embryo14. Here, we present a new technique to culture chick embryos ex ovo for high-resolution time-lapse imaging using transmitted light microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique (EC-culture)1 and allows for the culturing of chick embryos without the need for a climate chamber. The embryo is sandwiched between two identical filter paper carriers and is kept fully submerged in a simple, temperature-controlled medium covered by a layer of light mineral oil. Starting from the primitive streak stage (Hamburger-Hamilton stage 5, HH5)15 up to at least the 28-somite stage (HH16)15, embryos can be cultured with either their ventral or dorsal side up. This allows the acquisition of time-lapse movies covering about 30 hr of embryonic development. Representative time-lapse frames and movies are shown. Embryos are compared morphologically to an embryo cultured in the standard EC-culture.
The submerged filter paper sandwich provides a stable environment to study early dorsal and ventral morphogenetic processes. It also allows for live fluorescence imaging and micromanipulations, such as microsurgery, bead implantation, microinjection, gene silencing, and electroporation, and has a strong potential to be combined with immersion objectives for laser-based imaging (including light-sheet microscopy).

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Here, we present a new technique to culture chick embryos ex ovo for time-lapse imaging using transmitted light and fluorescence microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique1 that allows for the culturing of chick embryos fully-submerged in a liquid culture medium.

장기를위한 잠수 필터 종이 샌드위치<em&gt; 예 비켜</em초기 병아리 태아의&gt; 시간 경과 영상

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of ...

Induction of Hypoxia in Living Frog and Zebrafish Embryos

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and zebrafish embryos. Our system comprises a chamber featuring a simple setup that is nevertheless robust to induce and maintain a specific oxygen concentration and temperature in any experimental solution of choice. The presented system is very cost-effective but highly functional, it allows induction and sustainment of hypoxia for direct experiments in vivo and for various time periods up to 48 h.
To monitor and study the effects of hypoxia, we have employed two methods - measurement of levels of hypoxia-inducible factor 1alpha (HIF-1&#945;) in whole embryos or specific tissues and determination of retinal stem cell proliferation by 5-ethynyl-2&#8242;-deoxyuridine (EdU) incorporation into the DNA. HIF-1&#945; levels can serve as a general hypoxia marker in the whole embryo or tissue of choice, here embryonic retina. EdU incorporation into the proliferating cells of embryonic retina is a specific output of hypoxia induction. Thus, we have shown that hypoxic embryonic retinal progenitors decrease proliferation within 1 h of incubation under 5% oxygen of both frog and zebrafish embryos.
Once mastered, our setup can be employed for use with small aquatic model organisms, for direct in vivo experiments, any given time period and under normal, hypoxic or hyperoxic oxygen concentration or under any other given gas mixture.

We introduce a novel hypoxic chamber system for use with aquatic organisms such as frog and zebrafish embryos. Our system is simple, robust, cost-effective and allows the induction and sustainment of hypoxia in vivo and for up to 48 h. We present 2 reproducible methods to monitor the effectiveness of hypoxia.

살아있는 개구리와 Zebrafish 태아의 저산소증 유도

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and ...

Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to numerous cell types throughout the body. Understanding the biology of the neural crest has been limited, reflecting a lack of genetically tractable models that can be studied in vivo and in real-time. Zebrafish is a particularly important developmental model for studying migratory cell populations, such as the neural crest. To examine neural crest migration into the developing eye, a combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time videos of the developing eye in transgenic zebrafish embryos, namely Tg(sox10:EGFP) and Tg(foxd3:GFP), as sox10 and foxd3 have been shown in numerous animal models to regulate early neural crest differentiation and likely represent markers for neural crest cells. Multi-photon time-lapse imaging was used to discern the behavior and migratory patterns of two neural crest cell populations contributing to early eye development. This protocol provides information for generating time-lapse videos during zebrafish neural crest migration, as an example, and can be further applied to visualize the early development of many structures in the zebrafish and other model organisms.

A combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time imaging of neural crest migration in Tg(sox10:EGFP) and Tg(foxd3:GFP) zebrafish embryos.

실시간으로 개발을 시각화 하기 위해 다중 광자 시간 경과 영상: 마이그레이션 Zebrafish 태아 신경 크레스트 셀의 시각화

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to ...

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells (HSPCs) in Developing Zebrafish Embryos

Hematopoiesis is an essential cellular process in which hematopoietic stem and progenitor cells (HSPCs) differentiate into the multitude of different cell lineages that comprise mature blood. Isolation and identification of these HSPCs is difficult because they are defined ex post facto; they can only be defined after their differentiation into specific cell lineages. Over the past few decades, the zebrafish (Danio rerio) has become a model organism to study hematopoiesis. Zebrafish embryos develop ex utero, and by 48 h post-fertilization (hpf) have generated definitive HSPCs. Assays to assess HSPC differentiation and proliferation capabilities have been developed, utilizing transplantation and subsequent reconstitution of the hematopoietic system in addition to visualizing specialized transgenic lines with confocal microscopy. However, these assays are cost prohibitive, technically difficult, and time consuming for many laboratories. Development of an in vitro model to assess HSPCs would be cost effective, quicker, and present fewer difficulties compared to previously described methods, allowing laboratories to quickly assess mutagenesis and drug screens that affect HSPC biology. This novel in vitro assay to assess HSPCs is performed by plating dissociated whole zebrafish embryos and adding exogenous factors that promote only HSPC differentiation and proliferation. Embryos are dissociated into single cells and plated with HSPC-supportive colony stimulating factors that cause them to generate colony forming units (CFUs) that arise from a single progenitor cell. These assays should allow more careful examination of the molecular pathways responsible for HSPC proliferation, differentiation, and regulation, which will allow researchers to understand the underpinnings of vertebrate hematopoiesis and its dysregulation during disease.

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells HSPCs in Developing Zebrafish Embryos

Here, we present a simple method to quantitate hematopoietic stem and progenitor cells (HSPCs) in embryonic zebrafish. HSPCs from dissociated zebrafish are plated in methylcellulose with supportive factors, differentiating into mature blood. This allows the detection of blood defects and allows drug screening to be easily conducted.

조 혈 줄기와 뿌리를 Quantitate를 생체 외에서 시험의 개발 세포 (HSPCs) Zebrafish 태아를 개발

Hematopoiesis is an essential cellular process in which hematopoietic stem and progenitor cells (HSPCs) differentiate into the multitude of different cell ...

Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in zebrafish during different developmental stages. To investigate the role of endoglin(eng) in vessel formation during the development of hereditary hemorrhagic telangiectasia&#160;(HHT), morpholino-mediated targeted knockdown of eng in zebrafish are used to study its temporal expression and associated functions. Here, whole-mount in situ RNA hybridization (WISH) is employed for the analysis of eng and its downstream genes in zebrafish embryos. Also, tube formation assays are performed in HHT patient-derived induced pluripotent stem cell-differentiated&#160;endothelial cells (iPSC-ECs; with eng mutations). A specific signal amplifying system using the whole amount In Situ Hybridization &#8211; WISH provides higher resolution and lower background results compared to traditional methods. To obtain a better signal, the post-fixation time is adjusted to 30 min after probe hybridization. Because fluorescence staining is not sensitive in zebrafish embryos, it is replaced with diaminobezidine (DAB) staining here. In this protocol, HHT&#160;patient-derived iPSC lines containing an eng mutation are differentiated into endothelial cells. After coating a plate with basement membrane matrix for 30 min at 37 &#176;C, iPSC-ECs are seeded as a monolayer into wells and kept at 37 &#176;C for 3 h. Then, the tube length and number of branches are calculated using microscopic images. Thus, with this improved WISH protocol, it is shown that reduced eng expression affects endothelial progenitor formation in zebrafish embryos. This is further confirmed by tube formation assays using iPSC-ECs derived from a patient with HHT. These assays confirm the role for eng in early vascular development.

Presented here is a protocol for whole-mount in situ RNA hybridization analysis in zebrafish and tube formation assay in patient-derived induced pluripotent stem cell-derived endothelial cells to study the role of endoglin in vascular formation.

혈관 발달에서 엔도글린의 역할을 연구하기 위해 iPSC-ECs의 제브라피시 배아 및 튜브 형성 분석에서 시투 혼성화에 대한 전체 마운트

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in ...

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs during early development are technically challenging due to the limiting cell number. Moreover, many approaches require a miRNA of interest to be defined in assays such as northern blotting, microarray, and qPCR. Therefore, the expression of many miRNAs and their isoforms have not been studied during early development. Here, we demonstrate a protocol for small RNA sequencing of sorted cells from early mouse embryos to enable relatively unbiased profiling of miRNAs in early populations of neural crest cells. We overcome the challenges of low cell input and size selection during library preparation using amplification and gel-based purification. We identify embryonic age as a variable accounting for variation between replicates and stage-matched mouse embryos must be used to accurately profile miRNAs in biological replicates. Our results suggest that this method can be broadly applied to profile the expression of miRNAs from other lineages of cells. In summary, this protocol can be used to study how miRNAs regulate developmental programs in different cell lineages of the early mouse embryo.

We describe a technique for profiling microRNAs in early mouse embryos. This protocol overcomes the challenge of low cell input and small RNA enrichment. This assay can be used to analyze changes in miRNA expression over time in different cell lineages of the early mouse embryo.

초기 마우스 배아에서 시퀀싱을 위한 작은 RNA 라이브러리 의 준비

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs ...

A Simple and Effective Transplantation Device for Zebrafish Embryos

Classical embryological manipulations, such as removing cells and transplanting cells within or between embryos, are powerful techniques to study complex developmental processes. Zebrafish embryos are ideally suited for these manipulations since they are easily accessible, relatively large in size, and transparent. However, previously developed devices for cell removal and transplantation are cumbersome to use or expensive to purchase. In contrast, the transplantation device presented here is economical, easy to assemble, and simple to use. In this protocol, we first introduce the handling of the transplantation device as well as its assembly from commercially and widely available parts. We then present three applications for its use: generation of ectopic clones to study signal dispersal from localized sources, extirpation of cells to produce size-reduced embryos, and germline transplantation to generate maternal-zygotic mutants. Finally, we show that the tool can also be used for embryological manipulations in other species such as the Japanese rice fish medaka.

Embryological manipulations such as extirpation and transplantation of cells are important tools to study early development. This protocol describes a simple and effective transplantation device to perform these manipulations in zebrafish embryos.

Zebrafish 배아를 위한 간단하고 효과적인 이식 장치

Classical embryological manipulations, such as removing cells and transplanting cells within or between embryos, are powerful techniques to study complex ...