The authors would like to acknowledge Dr. Robert Price and the staff of the Instrumentation Resource Facility at the University of South Carolina School of Medicine. This work was partially supported by a SPARC Graduate Research Grant from the Office of the Vice President for Research at the University of South Carolina (JDP/VM). In addition this work was supported by Cook Biotech research agreement (JDP) and by FirstString Research Inc (JDP) and NIH 2 P20-RR016434-06 (JDP). In addition, NIH IDeA Networks of Biomedical Research Excellence (INBRE) grant for South Carolina P20GM103499 (JE)

조류 배아는 IUCAC의 규정에 따라 척추 동물로 간주되지 않습니다. 1. 수술에 대한 배아를 얻기 <ol><li> 80 품어 - 해밀턴과 햄버거 (HH) 단계에서 배아를 얻기 위해 약 72 시간 동안 40 ° C에서 가습 (60 %) 로커 배양기에서 90 수정 된 Bovan 닭고기 달걀 (무딘 끝을 위로하는) (17)에 기반 계란의 정확한 수를 결정 전력 분석 및 배아 (17)의 생존율. 적절한 공기 순환을 허용하도록 배양 플라스틱 계란 트레이를 사용합니다. </li><li> 이 원하는 발달 단계가 달성되면, 100mm X 26mm 페트리 접시에 약 20 ㎖ (중탄산 나트륨 (1g / L)로 보충) 따뜻한 (37 ° C) 타이로드의 버퍼를 붓는다. </li><li> 70 % 에탄올로 달걀 껍질을 소독. </li><li> 조심스럽게 메스 손잡이와 쉘 균열 조심스럽게 타이로드의 버퍼를 포함하는 접시에 그 내용을 공개. 그들은 시각적으로 표시하는 경우 배아 (i)를 폐기 이상 (ⅱ) 그들은오른쪽 발달 단계에서 (ⅲ) 그들은 잘못 노른자 및 / 또는 (ⅳ) 출혈 발생의 방향되지 않습니다. 즉시 개발을 손상하지 않도록 40 ​​° C에서 비켜 전 배치 후 수술을 실시되지 않은 배아를 유지합니다. 주 : 준비는 해밀턴과 햄버거 (18)에 따라 수행된다. 병아리 태아의 이상은 육안에 의해 절개 범위에 따라 결정된다. 배아는 적절한 폴딩 및 벤딩과 혈관이 있는지 확인합니다. </li></ol> 2. OFT 밴딩 <ol><li> 하나의 11/0 나일론 수술 봉합사에서 개별 1cm 긴 스레드를 Tweeze과 느슨한 매듭을 형성​​한다. 모든 예비 성형 노트를 살균 UV. </li><li> 해부 현미경, 시각적으로 심장이 정상 속도 (~ 120 비트 / 분)에 치고되어 있는지 확인합니다. 그렇지 않은 경우, 배아를 버린다. </li><li> 전송 피펫을 사용하여 배아 표면에 따뜻한 (37 ° C) 타이로드의 버퍼 6 ml의 - 그냥 수술 전, 5를 적용합니다. </li><li> , 공정 거래 청에서 예비 성형 매듭의 한 끝을 통과 OFT / 심실 접합 (OVJ) (또는 원하는 위치)에서 봉합사를 놓고하여 심장을 둘러싸고있는 세포막을 포함하여 OFT을 죄기 매듭으로 끝을 전달합니다. </li><li> 따뜻한 6 ㎖ (37 ° C) 타이로드의 버퍼가 전송 피펫을 사용하여 - 수술 후 5 노른자의 표면을 젖은. </li><li> 제어 태아에게 줄무늬 배아 등 전 비켜을 유지; 그러나 수술에 대상이되지 않습니다. </li><li> 습윤 (60 %) 인큐베이터에서 40 ℃에서 시간의 소정 시간 동안, 배아를 인큐베이션. </li><li> 원하는 시점에 도달하면, 직선 위를 이용 난황에서 배아를 절제. 미세 집게로 심장을 해부 등으로 인해 변경된 혈역학 17 심장 형태의 변화로 여러 하류 연구에 사용합니다. </li></ol> 3. 밴딩 개입이 혈류의 변화를 원인 것을 확인 주 : 밴딩 개입에 의한 부분적인 수축이 OVJ에서 혈류 속도의 증가를 이끈다. 이는 혈역학 파라미터 편리 실험의 목적하는 시점에서 수행되는 2 차원 초음파 영상을 이용하여 평가된다. <ol><li> 단일 배아 한번에 이미징 될 수 있기 때문에, 40 ° C의 배양기에서 초음파 영상에 대해 지정된 다른 배아를 유지한다. </li><li> 배아를 포함하는 배양 접시 40 ° C로 설정 가열 패드에 몇 군데에 배치합니다. </li><li> 따뜻한 (37 ° C) 타이로드의 버퍼로, 가장자리까지 접시를 입력합니다. 그대로 노른자를 유지하기 위해 천천히 접시에 완충 용액을 붓는다. 그러나, 난황의 무결성이 단계 동안 손상되​​는 경우, 배아를 버린다. </li><li> 배아의 중심 축이 조절 스탠드에서 일시 중단되는 초음파 프로브에 수직 인 것을 동양 배아 등. </li><li> 초음파 시스템 오페라팅은 B-모드에서 화면의 박동 심장의 2D 이미지를 획득하고 OFT, 심실과 OVJ 명확하게 볼 수 있습니다 있도록 스테이지 (손 난로)를 이동합니다. </li><li> 원하는 펄스 반복 주파수 (예를 들어, 20 kHz에서)에서, 펄스 파 (PW) 모드로 전환하고 OVJ 정확히 속도 데이터를 얻을 수 있습니다. 심박동의 B 모드 영상이 화면에 얻어진다. <ol><li> 공정 거래 청, 심실과 OVJ을 볼 수있는 무대를 이동합니다. 초음파 시스템 소프트웨어를 사용하여 정확하게 OVJ로 속도 측정을 얻었다. </li></ol></li></ol> 주 : 배아의 심장 박동수는 촬상 동안 감소하면, 얻어진 속도 데이터를 분석을 위해 사용되지 않아야한다. 속도 측정에 사용 된 모든 배아는 바람직하게는 초음파 촬상 후에 다른 실험에 사용되어서는 안된다.

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The authors have nothing to disclose.

이 기술은 상대적으로 빠르게 수행하기 쉬운, 그러나 특정 키 포인트는 정확한 다운 스트림 결과를 얻을 수 있도록 염두에 보관해야합니다. 배아는 충분한 수분 보충을 제공하는 타이로드의 버퍼를 포함하는 배양 접시에서 비켜 예를 유지되어야한다. 타이로드의 버퍼와 노른자 수술 후 수분을하고 배양 챔버가 적절하게 수화되어 있는지 확인하는 것도 중요하다. 출혈이 볼 수 또는 노른자가 조금이라도 깨진 경우 수술은 배아에서 수행 할 수 없습니다. 특히 장기간의 실험, 배아의 생존에 영향을 미칩니다. 배아 치사의 또 다른 잠재적 인 원인은, 첫째, 따라서 크게 심장 기능을 손상시키지 적용 할 때 OFT 주위에 밴드가 너무 꽉 여부입니다. 현재, 우리는 OFT 밴딩에 혈류 속도의 변화를 결정하는 도구로서 확증 초음파를 사용한다. 미래에, 우리는 (마이크로 컴퓨터 단층 촬영을 이용하여 확인합니다CT). 우리는 또한 시각적으로 B 모드 2D 이미지에서 OFT 주위의 밴드 압박감을 결정합니다. 중요한 것은, 심장 박동 및 체적 흐름 (CFD를 사용하여 계산)의 차이는 줄무늬와 대조군 17 배아 사이에 관찰되지 않습니다. 이들은 심장 혈관 시스템의 생리 따라서 더 밴딩 개입의 유효성을 검사의 줄무늬 배아에 손상되지 않았 음을 나타냅니다. 목적하는 시점에서, 밴딩 후, 분석은 여러 중요한 밸브 발달 과정에서 혈역학 변경의 효과를 이해하기 위해 수행 될 수있다. 이들은 다음을 포함하지만, ECM 성분들의 위치 파악을 결정하고 OFT의 미세 구조를 결정하고, 밸브의 개발에 관련된 핵심 조절 유전자의 전사 수준의 변화를 결정하고, 이에 제한되지 않는다. 마지막으로,이 기술은 윈도우 (19) 내부 계란 배아에서 OFT 밴딩의 현재 방법을 개선한다. 그 방법은 심각LY 하류 분자 분석을 수행 할 필요가 배아를 축적하는 무능력에 의해 방해. 이 새로운 기술은 물론 구조 실험에 사용될 수있다. OFT 줄무늬가 구조 및 배아가 모욕을 복구하도록 허용하는이 응용 프로그램은 수 있습니다. 전류 메소드는 수행하지 여전히 유의 하류 분석을 수행 할 수있다. 우리는 성공적으로 초기 발달 단계 (- 17 HH 16)에 병아리 태아의 심장에 혈류 역학적 자극을 변경하려면이 기술을 사용하고있다. 발달의 후기 단계에서 수술을 수행 할 때 밴딩 프로토콜 인해 배외 (extraembryonic) 혈관의 어느 새김의 위험에 도전을 제공 할 수 있습니다. 또한, 개발의 후반 단계에서, 계란 내용이 배양 접시에 옮겨 때 그대로 골목 노른자를 유지하는 것이 어렵다.

Abnormally formed outflow valves are the most common type of congenital heart defects 1. However, defective cardiac valve structure and function, even though present at birth, may become symptomatic only in adulthood. In fact, several adult valve diseases can be attributed to a congenital origin. Treatment of such patients often involves replacing defective valves, and, importantly, replaced aortic valves have been shown to have congenital anomalies 2. Given the fact that critical processes involved in valve development begin early during embryogenesis, the importance of better understanding the mechanisms that regulate these events is highlighted. 
The primitive heart tube, which is the first functioning organ in an embryo, exhibits two distinct layers - an endothelial endocardium surrounded by myocardium - separated by extracellular matrix (cardiac jelly) which is mostly produced and secreted by the myocardium 3-5. As development continues, valve primordia (endocardial cushions) are formed, after rightward looping of the embryonic heart, by local expansion of the cardiac jelly at the atrioventricular (AV) canal and the outflow tract (OFT) 4,6. This expansion is mediated by the highly regulated process of epithelial-mesenchymal transition (EMT), during which the cardiac jelly becomes populated by endocardially-derived mesenchymal cells 6. In addition to the mesenchymal population derived through EMT, neural crest cells are also involved in valvulogenesis of the OFT 3. 
Hemodynamic stimuli, such as shear stress, are important epigenetic factors that regulate heart development in the embryo 7,8. Using a 3D in vitro system, we have previously shown shear stress to be a factor influencing the expression and deposition of fibrous extracellular matrix (ECM) proteins in AV and OFT cushions 9,10. Moreover, studies carried out by several researchers have demonstrated that altered blood flow leads to improper valves and septa formation 11-16. Recently, using the novel banding procedure presented here, we have shown that changing hemodynamics in the embryonic chick heart affects the early processes involved in OFT valve formation 17.
The technique described here provides a novel model for altering hemodynamics in the developing chick heart by partially constricting the OFT ex ovo. This reproducible procedure is relatively quick and allows researchers to obtain a sufficient number of embryos/whole hearts/OFT tissue, etc. for downstream analyses including gene expression studies. Moreover, this new model can be used to study 'chronic' effects of altered hemodynamics on OFT valve development.

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			<td height="105" style="height:105px;width:216px;">Fertilized Bovan chicken eggs</td>
			<td style="width:71px;">Clemson University, Clemson, SC</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">11/0 Nylon suture</td>
			<td style="width:71px;">Ashaway</td>
			<td style="width:119px;">S30001</td>
			<td>UV sterilize knots before surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">100 x 26 mm petri dish</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">25387-030</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Transfer pipettes</td>
			<td style="width:71px;">Thermo Scientific&#160;</td>
			<td style="width:119px;">232-20S</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Scalpel handle #3</td>
			<td style="width:71px;">Fine Science Tools</td>
			<td style="width:119px;">91003-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Straight scissor</td>
			<td style="width:71px;">Roboz</td>
			<td style="width:119px;">RS-6702</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Dumont #5 fine forceps</td>
			<td style="width:71px;">Fine Science Tools</td>
			<td style="width:119px;">11254-20</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tyrodes buffer</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">2145-10L</td>
			<td>Filter sterlize before use&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sodium bicarbonate</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">S233-500</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Vevo 770 Ultrasound Imaging system</td>
			<td style="width:71px;">VisualSonics, Inc.</td>
			<td style="width:119px;">VS-11392</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">708 Ultrasound transducer&#160;</td>
			<td style="width:71px;">VisualSonics, Inc.</td>
			<td style="width:119px;">VS-11171</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

그림 1은 OFT 밴딩에 필요한 악기를 사용하는 것이 좋습니다. 뚜껑이 덮여 때 배아를 파괴하지 않도록 배아 예를 포함하는 페트리 접시 (도 1a)는 오보 충분히 깊어 야한다. 깊은 배양 접시 (도 1C)는 또한 타이로드의 버퍼의 충분한 양이 난황 위에 붓고 수 있도록 초음파 이미징을 위해 사용되어야한다. 단일 1cm 스레드 (그림 2A)는 11 / 0 나일론 봉합사 (그림 1A)로부터 tweezed과 느슨한 매듭 (그림 1B)로 연결되어 있습니다. 모든 예비 성형 노트는 UV 살균된다. 그림 3a는 수정 된 계란이 원하는 단계까지 배양 가습 로커 실을 보여줍니다. 그림 3b는 줄무늬 및 제어 배아 비켜 예를 유지 배아 인큐베이터를 보여줍니다 </e시간의 원하는 양 m&gt;. 두 인큐베이터 유형은 40 ° C로 유지된다. 도 4a에 도시 된 노른자위 예 오보.도 4b의 위쪽에 HH17 배아는 HH17 배아의 OVJ로 봉합 (밴드)의 위치를 개략적으로 보여주는 도면이다.도 4c는 묘화 줄무늬 배아의 대표 화상은 수술 후 48 시간. 함입 사이트를 통해 혈류 속도에 밴딩 간섭의 효과는 2D 초음파 영상에 의해 측정된다. 예상대로 OVJ의 속도 제어 (도 5)에 상대적인 밴드 배 높다. 최근 OFT 혈류 속도 및 벽 전단 응력 (17) 상에 줄무늬의 영향을 나타내는 24 시간 시점에서 줄무늬 제어 하트 전산 유체 역학 (CFD) 분석을 수행 하였다. &lt;유지-together.within 페이지 = &quot;1&quot;&gt; : FO P 클래스 = &quot;jove_content&quot; <img alt="그림 1" src="/files/ftp_upload/53955/53955fig1.jpg" /> 그림 1. 수술 도구. (A) 11/0 나일론 봉합사. (B) 미리 형성된 매듭을 포함하는 페트리 접시입니다. (C) 예 비켜 배아 문화와 초음파 영상에 대한 깊은 배양 접시. (D) 전송 피펫. (E) 메스는 달걀 껍질 균열 처리합니다. (F) 수술 후 연구를위한 배아 절단을위한 스트레이트 가위. (G) 미세 집게 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53955/53955fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 2" src="/files/ftp_upload/53955/53955fig2.jpg" /> OFT 밴딩 2. 봉합 그림. (A) 하나 1cm 스레드 봉합로부터 tweezed. <strong&gt; (B) 사전 형성 매듭. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53955/53955fig2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 3" src="/files/ftp_upload/53955/53955fig3.jpg" /> 3. 배양 챔버 그림. (A) 수정란을위한 가습 로커 인큐베이터. (B) 줄무늬 및 제어 배아의 문화 비켜 전을위한 가습 실. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53955/53955fig3large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 4" src="/files/ftp_upload/53955/53955fig4.jpg" /> 4. OFT 밴딩 그림. (A) 전체 HH17 병아리 배아는 수축을 묘사, 스케일 바 = 10mm (B) 도식 표현 예 비켜을 유지사이트 (빨간색). (C) 줄무늬 배아는 OFT 밴딩 후 48 시간 군데, 스케일 바 = 0.5 mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53955/53955fig4large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 5" src="/files/ftp_upload/53955/53955fig5.jpg" /> 그림 5. 속도 측정 초음파 유래. (A) 제어와 (B)의 대표 속도 프로파일은 1 시간 시점에서 얻은 배아를 줄무늬. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53955/53955fig5large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a>

Watch this Scientific Journal Video about A Novel Ex Ovo Banding Technique to Alter Intracardiac Hemodynamics in an Embryonic Chicken System at JoVE.com

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-novel-ex-ovo-banding-technique-to-alter-intracardiac-hemodynamics

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

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

6.5K 조회수.  School of Medicine, University of South Carolina. 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). 

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

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 ...

연구

발생학

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.

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 ...

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein, tandem dimer Tomato (tdTomato), are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons, evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations.

A protocol to generate a co-culture system consisting of neurons derived from induced pluripotent stem cells (iPSCs), primary cortical neurons and astrocytes is described. This co-culture system allows detection of the formation of synaptic contacts and circuits between new, iPSC-derived neurons and pre-existing cortical neurons expressing channelrhodopsin-2.

공동 문화 시스템에 신경 연결의 형성을 평가하기위한 Optogenetic 접근

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are ...

Generating CRISPR/Cas9 Mediated Monoallelic Deletions to Study Enhancer Function in Mouse Embryonic Stem Cells

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often located distally from the regulated gene in intergenic regions or even within the body of another gene. The position independent nature of enhancer activity makes it difficult to match enhancers with the genes they regulate. Deletion of an enhancer region provides direct evidence for enhancer activity and is the gold standard to reveal an enhancer's role in endogenous gene transcription. Conventional homologous recombination based deletion methods have been surpassed by recent advances in genome editing technology which enable rapid and precisely located changes to the genomes of numerous model organisms. CRISPR/Cas9 mediated genome editing can be used to manipulate the genome in many cell types and organisms rapidly and cost effectively, due to the ease with which Cas9 can be targeted to the genome by a guide RNA from a bespoke expression plasmid. Homozygous deletion of essential gene regulatory elements might lead to lethality or alter cellular phenotype whereas monoallelic deletion of transcriptional enhancers allows for the study of cis-regulation of gene expression without this confounding issue. Presented here is a protocol for CRISPR/Cas9 mediated deletion in F1 mouse embryonic stem (ES) cells (Mus musculus129 x Mus castaneus). Monoallelic deletion, screening and expression analysis is facilitated by single nucleotide polymorphisms (SNP) between the two alleles which occur on average every 125 bp in these cells.

Experimental validation of enhancer activity is best approached by loss-of-function analysis. Presented here is an efficient protocol that uses CRISPR/Cas9 mediated deletion to study allele-specific regulation of gene transcription in F1 ES cells which contain a hybrid genome (Mus musculus129 x Mus castaneus).

생성 CRISPR은 / Cas9 중재 Monoallelic 삭제할 경우는 마우스 배아 줄기 세포의 증강 기능을 연구하는

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often ...

Technique to Target Microinjection to the Developing Xenopus Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus embryos are large, develop externally, and can be easily manipulated by microinjection or surgical procedures. In addition, fate maps have been established for early Xenopus embryos. Targeted microinjection into the individual blastomere that will eventually give rise to an organ or tissue of interest can be used to selectively overexpress or knock down gene expression within this restricted region, decreasing secondary effects in the rest of the developing embryo. In this protocol, we describe how to utilize established Xenopus fate maps to target the developing Xenopus kidney (the pronephros), through microinjection into specific blastomere of 4- and 8-cell embryos. Injection of lineage tracers allows verification of the specific targeting of the injection. After embryos have developed to stage 38 - 40, whole-mount immunostaining is used to visualize pronephric development, and the contribution by targeted cells to the pronephros can be assessed. The same technique can be adapted to target other tissue types in addition to the pronephros.

Technique to Target Microinjection to the Developing Xenopus Kidney

Here, we present a protocol to use fate maps and lineage tracers to target injections into individual blastomeres that give rise to the kidney of Xenopus laevis embryos.

개발에 미세 주입을 대상으로 기술<em&gt; Xenopus의</em&gt; 신장

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. The inner ear auditory sensory organ is critical for our ability to hear. It houses a highly specialized sensory epithelium that consists of mechano-transducing hair cells (HCs) and surrounding glial-like supporting cells (SCs). Despite structural differences in the auditory organs, molecular mechanisms regulating the development of the auditory organ are evolutionarily conserved between mammals and aves. In ovo electroporation is largely limited to early stages at E1 - E3. Due to the relative late development of the auditory organ at E5, manipulations of the auditory organ by in ovo electroporation past E3 are difficult due to the advanced development of the chicken embryo at later stages. The method presented here is a transient gene transfer method for targeting genes of interest at stage E4 - E4.5 in the developing chicken auditory sensory organ via in ovo micro-electroporation. This method is applicable for gain- and loss-of-functions with conventional plasmid DNA-based expression vectors and can be combined with in ovo cell proliferation assay by adding EdU (5-ethynyl-2&#180;-deoxyuridine) to the whole embryo at the time of electroporation. The use of green or red fluorescent protein (GFP or RFP) expression plasmids allows the experimenter to quickly determine whether the electroporation successfully targeted the auditory portion of the developing inner ear. In this method paper, representative examples of GFP electroporated specimens are illustrated; embryos were harvested 18 - 96 hr&#160;after electroporation and targeting of GFP to the pro-sensory area of the auditory organ was confirmed by RNA in situ hybridization. The method paper also provides an optimized protocol for the use of the thymidine analog EdU to analyze cell proliferation; an example of an EdU based cell proliferation assay that combines immuno-labeling and click EdU chemistry is provided.

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

The auditory organ mediates hearing. Here we present a modified in ovo micro-electroporation method optimized for studying auditory progenitor cell proliferation and differentiation in the developing chicken auditory organ.

치킨 청각 기관에 유전자 전달에 의한<em&gt; 비켜에서</em&gt; 마이크로 전기

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. ...

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 ...

In Ovo Electroporation in the Chicken Auditory Brainstem

Electroporation is a method that introduces genes of interest into biologically relevant organisms like the chicken embryo. It is long established that the chicken embryo is an effective research model for studying basic biological functions of auditory system development. More recently, the chicken embryo has become particularly valuable in studying gene expression, regulation and function associated with hearing. In ovo electroporation can be used to target auditory brainstem regions responsible for highly specialized auditory functions. These regions include the chicken nucleus magnocellularis (NM) and nucleus laminaris (NL). NM and NL neurons arise from distinct precursors of rhombomeres 5 and 6 (R5/R6). Here, we present in ovo electroporation of plasmid-encoded genes to study gene-related properties in these regions. We show a method for spatial and temporal control of gene expression that promote either gain or loss of functional phenotypes. By targeting auditory neural progenitor regions associated with R5/R6, we show plasmid transfection in NM and NL. Temporal regulation of gene expression can be achieved by adopting a tet-on vector system. This is a drug inducible procedure that expresses the genes of interest in the presence of doxycycline (Dox). The in ovo electroporation technique &#8211; together with either biochemical, pharmacological, and or in vivo functional assays &#8211; provides an innovative approach to study auditory neuron development and associated pathophysiological phenomena.

In Ovo Electroporation in the Chicken Auditory Brainstem

Auditory brainstem neurons of avians and mammals are specialized for fast neural encoding, a fundamental process for normal hearing functions. These neurons arise from distinct precursors of embryonic hindbrain. We present techniques utilizing electroporation to express genes in the hindbrain of chicken embryos to study gene function during auditory development.

닭 오라클 Brainstem의 Ovo Electroporation에서

Electroporation is a method that introduces genes of interest into biologically relevant organisms like the chicken embryo. It is long established that ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

혈관 내피 줄기 세포의 혈관 생성을 시각화하기위한 유방 팻 패드 이식 기술

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ideal for experimentation. Currently, the study of these developmental pathways in the chicken embryo is conducted by applying inhibitors and drugs at localized sites and at low concentrations using a variety of methods. In vitro tissue&#160;culturing is a technique that enables the study of tissues separated from the host organism, while simultaneously bypassing many of the physical limitations present when working with whole embryos, such as the susceptibility of embryos to high doses of potentially lethal chemicals. Here, we present an organotypic culturing protocol for culturing the embryonic chicken half head in vitro, which presents new opportunities for the examination of developmental processes beyond the currently established methods.

Here, we present an organotypic culturing protocol to grow embryonic chicken organs in vitro. Using this method, the development of embryonic chicken tissue can be studied, while maintaining a high degree of control over the culture environment.

Organotypic 문화 메서드 배아 치킨 조직 개발 연구를

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ...