저자는 프로토콜의 개발에 그의 예비 작업에 대 한 그레고리 할 러 (마운트 세인트 빈센트 대학)를 감사 하 고 싶습니다. 저자 그의 기술적 전문성과 촬영 및 원고에 대 한 오디오/비디오 부분의 생산에 대 한 니콜라스 존스 (마운트 세인트 빈센트 대학)를 감사 하 고도 싶습니다. 다니엘 앤드류 스에서 MSVU 및 자연 과학 캐나다 엔지니어링 연구 위원회 (NSERC) 통해 학부 학생 연구 보너스 자금에 의해 지원 되었다. 타마라 A. 프란츠-Odendaal NSERC 발견 교부 금에 의해 지원 됩니다.

참고: 배아 단계에 대 한 햄버거와 해밀턴12 (HH) 준비 테이블을 사용 합니다.
1. 배아의 부 화
<ol><li>37 ° C ± 1 ° C, 40% 습도에서 살 균, 온도 제어 인큐베이터에서 수정 된 닭 알을 품 어.</li>
	<li>계란 1 차례 하루 x HH34을 알을 품을 수 있도록 (8 일 후 수정).</li>
</ol>2. 준비 및 재료의 살 균
<ol><li>12 배아, 압력솥 증류수, 0.85% 병아리 염 분, 12 유리 펫, 종이 조직과 24 2.5 c m x 2.5 c m 사각 반 다공성 필터 종이, 강선의 24 2.5 c m x 2.5 c m 사각형의 1 상자 1 L 2 L 메쉬 곡선 아래쪽 가장자리에 대 한. 자료, 소독 압력솥 적어도 15 분 동안 121 ° C에서 그들. 참고: 재료의 압력가 마로 소독 사전에 할 수 있습니다.</li>
	<li>측면, 선반, 및 문을 닦아 하 여 70% 에탄올과 인큐베이터를 소독. 장소 2 플라스틱 용기 포함 압력가 하는 습도 유지 하기 위해 인큐베이터 안에 증류수. 37 ° C ± 1 ° C, 40% 습도에 문화 인큐베이터 prewarm 인큐베이터는 완전히 어두운; 확인 문은 투명, 은박지와 커버.</li>
	<li>문화 인큐베이터에 그들을 배치 하 여 영양소 매체의 및 페니실린의 1 mL 100 mL를 prewarm.</li>
	<li>종이 수건으로 작업 영역을 커버 하 고 70% 에탄올을 분사 하 여 벤치를 소독. 비슷한 방식으로 소독 집게, 해 부가 위, 면도날, 및 70% 에탄올과 플라스틱 숟가락의 2 쌍.</li>
	<li>장소 12 살 균 100 mm 페 트리 접시 그리고 사용에 대 한 벤치에 접시 24 살 균 35 m m. 요리 사용까지 멸 균 가방에 남아 확인 합니다.</li>
</ol>3. 배아 준비
참고:이 시점에서 앞으로, 문화를 훼손 하지 않도록 보호 먼지 얼굴 마스크를 착용 하십시오. 세균 이나 곰 팡이 감염 문화를 망칠 것입니다 그리고 그것은 인큐베이터에서 모든 문화에 빠르게 확산의 위험.
<ol><li>균열 계란 열고 100 mm 페 트리 접시 0.85% 병아리 염 분을 포함 하는 배아를 전송 합니다. 해 부 특징에 따라 배아 햄버거와 해밀턴 준비 지침12 에서 설명 하 고 태아를 확인 단계는 HH34에서 이다.</li>
	<li>목 고 소독된 면도날을 사용 하 여 중간에 태아의 머리를 이등분. 소독 집게를 사용 하 여 두뇌를 제거 합니다. 부리는 그대로 둡니다.</li>
</ol>4. 문화 설치
<ol><li>한 100 mm 페 트리 접시 안에 35 mm 페 트리 접시의 장소 2.</li>
	<li>각 35 mm 페 트리 접시 안에 강철 와이어 메쉬를 배치 합니다.</li>
	<li>집게를 사용 하 여 전송 bisected 머리의 1 2.5 c m x 2.5 c m 반 다공성 필터 종이 위로 향하도록 눈으로. 조직과 보안은 약간 그것을 기울이기로 벗어 버리는 하지 않습니다 확인 합니다.</li>
	<li>집게를 사용 하 여 신중 하 게 장소 눈 조직 및 필터 종이 위에 올려진된 무대 위에 눈 조직으로 만드는 35mm 페 트리 요리의 1 강철 와이어 메쉬.</li>
	<li>4.3 및 4.4 다른 절반 머리와 단계를 반복 합니다.</li>
	<li>필터 종이의 수준에 도달할 때까지 각 35 mm 페 트리 접시에 직접 영양소 매체를 추가 신중 하 게 메 마른 유리 피 펫을 사용 하 여. 영양소 중에 반 머리를 잠수함 하지 마십시오.</li>
	<li>각 페 트리 접시에 영양 보통 10000 U/mL 페니실린-스의 50 µ L를 추가 합니다.</li>
	<li>작은 광장으로 조직 종이 접어과 100 mm 페 트리 접시 안에 배치. 압력가 증류수와 함께 축 축 하 게.</li>
	<li>최대 4 일 동안 37 ° C ± 1 ° C, 40% 습도에서 살 균, 어두운 인큐베이터에 문화 접시를 놓습니다.</li>
	<li>각 태아에 대 한 3-4 단계를 반복 합니다. 참고: 배아의 수 특정 실험;에 따라 달라 집니다 필요 여기, 우리는 12 배아에 대 한 프로토콜을 설명합니다.</li>
</ol>5. 문화 유지 보수
<ol><li>하루에 한 번, 신선한, 압력가 증류수와 인큐베이터에서 플라스틱 용기를 가기.</li>
	<li>세균 이나 곰 팡이 감염에 대 한 모든 문화는 매일 확인 합니다. 문화는 붕괴 또는 매체 색상을 변경 되었습니다 감염 됩니다. 감염 되는 모든 문화를 삭제 합니다.</li>
</ol>6입니다. 고정
<ol><li>인큐베이터에서 모든 요리를 제거 하는 경작, 다음.</li>
	<li>부드럽게 집게의 쌍을 사용 하 여, 조직을 찢 어 하지 않도록 주의 복용 필터 종이에서 눈을 제거 합니다.</li>
	<li>4 ° C에서 하룻밤 염 인산 염 버퍼 x 1 또는 10% 중립 버퍼링 말린 하룻밤 실 온에서 4 %paraformaldehyde 눈 조직을 수정 했다.</li>
	<li>인산 염 버퍼 염 분 x 1에 4 ° C에서 티슈를 저장 합니다.</li>
</ol>

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저자는 공개 없다.

이 프로토콜 닭 눈 배아 주 8에서에서 (HH34)의 성장을 달성 하기 위해 설립 된 조직 배양 기술을 사용 생체 외에서 4 일 동안. 이 그리드 경작 방법 Trowell15에 의해 원래 기술 되었다. 우리는 핀 토와 홀의 1991 연구16 배아 닭 눈15의 분리 된 조직 층 사이 유도 신호를 공부 하는 그리드와 반 다공성 멤브레인을 활용 하 여 프로토콜을 최적화. ?...

배아 닭고기 (갈 루스 갈 루스)는 생물학의 분야에서 일반적으로 사용 되는 우수한 모델 유기 체. 그 잠복기는 대략 21 일 고 많은 계란 수 있습니다 알을 품을 동시에, 신속 하 고 효율적인 실험을 만들기. 아마도 가장 중요 한 것은, 태아는 또한 쉽게 조작, 광범위 한 연구와 유전자와 이러한 프로세스를 구동 하는 단백질의 주요 개발 프로세스의 사용.
미 발달 닭 눈은 개발을 통해 다양 한 다른 조직 비슷한 많은 다른 시체는 시스템의 상호 작용 하는 복잡 한 기관 이다. 이 메서드는 개발의 고급 단계에서 특히이 조직 개발의 연구를 수 있습니다. 예를 들어 다층된 망막 신경 시스템의 개발을 공부 하 고 그에 게 특히 관심이 될 수 있습니다. 와 같은 각 막, vitreal, 렌즈, sclera, 몸과 눈 꺼 풀 연구자에 게 도움이 다른 눈 조직의 연구를 활성화 하는 방법에 대 한 대체. 닭 배아 눈에는 편평한 뼈, intramembranous 뼈 유도 및 척추1에서 나오고의 연구에 대 한 모델로 사용 될 수 있는 scleral 이소골의 일련을 포함 되어 있습니다.
현재, 배아 발달을 공부 하는 데 사용 하는 방법 수가 있다. 억제 항 체 또는 다른 억제 분자2,3, microinjections 수술 microbeads 억제 물4에 배어 이식 및 electroporation5 는 downregulate 유전자를 사용할 수 있는 모든 방법을 또는 태아의 단백질입니다. 비슷한 메서드는 upregulate 단백질에 사용 됩니다. 이러한 방법을 그들의 제한 없이 되지 않습니다. 예를 들어 화학 물질을 사용 하 여 변경 하는 배아 개발을 태아에 치명적인 영향을 평가 해야 합니다, 그리고 이것의 생존을 보장 하기 위해 충분히 낮은 복용량에서 응용 프로그램의 지역화 된 사이트에 앞서 언급 한 방법의 사용을 제한 합니다 태아입니다.
생체 외에서 조직 배양 연구 개발 생물의 넓은 범위에서 사용 되었습니다 그리고 상기 제한 중 일부를 무시 하는 데 사용할 수 있습니다. 예를 들어 femora6, 깃털 꽃 봉 오리7,8및 사지9 닭의 모든 되었습니다 공부 마우스10 와 뿌리의 고환 및 식물11의 줄기 조직 배양 방법를 사용 하 여. 이러한 방법을 과학자 제어 온도 변동 양분 가용성을 변경 하는 등 조직 개발의 높은 학위를 부여 합니다. 전체 태아에서 직물의 절연은 또한 높은 농도에서 세계적인 규모에 대 한 조작 연구를 활성화 하는 화학 제품의 치명적인 효과를 훨씬 덜 취약 합니다. 체 외에서 배양 하는 또 다른 주목할 만한 이점은 이다 조직의 셀룰러 환경 보존 조직 배열 다른 조직 유형9사이의 상호 작용을 공부 하는 게 가능 하, 상대적으로 변경 되지 않습니다. 따라서, 체 외에서 배양 열립니다 문을 추가 실험 접근 비켜 또는 vivo에서 모델에서 사용할 수 없습니다.
현재, 화학 물질을 사용 하 여 미 발달 닭 눈의 개발을 공부 하 고 특히 도전 이다. Extraembryonic 막 수 커버 태아, 어렵게 적용 microbeads 또는 화학 제품; 태아 그것 수록, 더는 이미 어려운 방법 복잡 계란 내에서 매우 적극적 이기도 합니다. 이 프로토콜에는 눈 및 그 주변 조직, 또한 눈 내 개발 프로세스를 검사 하는 새로운 기회를 제공 하면서이 방 벽을 제거 쉽게 액세스할 수 있습니다. 이 프로토콜은 배아 눈 내 scleral 이소골의 유도 연구 설립 되었다.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">35 mm cell culture petri dishes</td>
			<td style="width:71px;">Corning</td>
			<td style="width:119px;">353001</td>
			<td style="width:676px;">easy grip tissue culture dish, polystyrene, non-pyrogenic</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">100 mm cell culture petri dishes</td>
			<td style="width:71px;">Corning</td>
			<td style="width:119px;">353003</td>
			<td>tissue culture dish, polystyrene, non-pyrogenic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">paper tissue</td>
			<td style="width:71px;">Kimtech</td>
			<td style="width:119px;">34155</td>
			<td>Kimtech Science Brand Task Wipers, 280 per carton</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">wire mesh</td>
			<td style="width:71px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td>stainless steel wire mesh (grid size 0.7 mm)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">disposable glass pipettes</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">14673-010</td>
			<td>Borosilicate glass disposable 5 3/4&#34;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">nutrient medium</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">12591-038</td>
			<td>Fitton-Jackson Modification, [+] L-glutamine with phenol red (BGJB)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">penicillin-streptomycin</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">P4458</td>
			<td>10000 units/mL penicillin streptomycin solution stabilised</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">filter paper</td>
			<td style="width:71px;">Whatman</td>
			<td style="width:119px;">1454 090</td>
			<td>semi-porous filter paper 90mm</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:216px;">fertilized chicken eggs</td>
			<td style="width:71px;">Dalhousie University Agricultural College</td>
			<td style="width:119px;">n/a</td>
			<td>can be obtained from local farms</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">sodium chloride (NaCl)</td>
			<td style="width:71px;">EMD</td>
			<td style="width:119px;">SX0420-3</td>
			<td>sodium chloride crystals, reagent grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1 L glass bottle</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">89000-240</td>
			<td>1 L pyrex autoclavable glass bottle</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">ethanol</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td>BP82011</td>
			<td>70% molecular biology grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">tupperware containers</td>
			<td style="width:71px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td>store-bought and sterilized with EtOH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">disposable razor blades</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">55411-050</td>
			<td>single edge industrial razor blades (surgical carbon steel)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">plastic spoons</td>
			<td style="width:71px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td>store-bought and sterilized with EtOH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">dust mask</td>
			<td style="width:71px;">3M</td>
			<td style="width:119px;">n/a</td>
			<td>3M 8500 Comfort Mask</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">paraformaldehyde</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">P6148</td>
			<td>paraformaldehyde, reagent grade, crystalline</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">neutral-buffered formalin</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">72210</td>
			<td>10% neutral buffered formalin</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">phosphate buffered saline (PBS)</td>
			<td style="width:71px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td>10X phosphate buffered saline pH 7.4 (137mM NaCl, 2.5mM KCl, 4.3mM Na2HPO4, 1.4mM KH2PO4)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">15 ml falcon tubes</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">21008-216</td>
			<td>presterilized centrifuge tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">forceps</td>
			<td style="width:71px;">FST</td>
			<td style="width:119px;">n/a</td>
			<td>fine forceps</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">chick saline&#160;</td>
			<td style="width:71px;">n/a</td>
			<td style="width:119px;">n/a&#160;</td>
			<td>0.85% NaCl</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">tinfoil</td>
			<td style="width:71px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td>store-bought</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">paper towel</td>
			<td style="width:71px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td>store-bought</td>
		</tr>
	</tbody>
</table>

organotypic culture method to study the development of embryonic chicken tissues

미 발달 닭 척 추가 개발에 대 한 신뢰할 수 있는 모델 생물으로 주로 사용 됩니다. 접근성 및 짧은 인큐베이션 기간 적합 실험에 대 한 합니다. 현재, 닭 태아에 있는이 발달 경로 연구는 저 해제 및 지역화 된 사이트에서 다양 한 방법 사용 하 여 낮은 농도에서 마약을 적용 하 여 실시 됩니다. 생체 외에서 조직 배양은 조직 호스트 유기 체에서 동시에 전체 배아, 배아의 민감성 등을 작업할 때 현재 물리적 한계의 대부분을 무시 하면서 분리의 연구를 가능 하 게 하는 기술 잠재적으로 치명적인 화학 제품의 높은 복용량. 여기, 우리는 배아 치킨 반 머리 생체 외에서현재 설립된 방법 넘어 개발 프로세스의 시험에 대 한 새로운 기회를 선물 한다 경작을 위한 프로토콜을 경작 하는 organotypic 제시.

이 메서드를 사용 하 여 미 발달 닭 눈 수 수 경작 하루 8 개발 (HH34)의 생체 외에서 에서 4 일 동안. 비켜에 개발의 4 일 HH38에 해당 합니다.
이 자란 방법 깃털 싹 눈 주위와 눈 꺼 풀 (그림 1B)에 개발을 지원 합니다. 이러한 깃털 싹 존재 비켜 HH34에서 경작 (그림 1A),...

Watch this Scientific Journal Video about Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues at JoVE.com

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

Organotypic 문화 메서드 배아 치킨 조직 개발 연구를

여기, 선물이 배아 치킨 장기 성장 프로토콜을 경작 하는 organotypic 생체 외에서. 이 메서드를 사용 하 여 미 발달 닭 조직 개발 수 수 공부, 문화 환경에 통제의 고차를 유지 하면서.

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

organotypic-culture-method-to-study-development-embryonic-chicken

Research

JoVE Journal

Developmental Biology

5.9K 조회수.  Mount Saint Vincent University. 여기, 선물이 배아 치킨 장기 성장 프로토콜을 경작 하는 organotypic 생체 외에서. 이 메서드를 사용 하 여 미 발달 닭 조직 개발 수 수 공부, 문화 환경에 통제의 고차를 유지 하면서.

비디오: Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair cells are aligned in one row of inner hair cells and three rows of outer hair cells that extend along the basal to apical axis of the cochlea. The explant culture technique described here provides an efficient method to isolate and maintain cochlear explants from the embryonic mouse inner ear. Also, the morphology and molecular characteristics of sensory hair cells and nonsensory supporting cells within the cochlear explant cultures resemble those observed in vivo and can be studied within its intrinsic cellular environment. The cochlear explants can serve as important experimental tools for the identification and characterization of molecular and genetic pathways that are involved in cellular specification and patterning. Although transgenic mouse models provide an effective approach for gene expression studies, a considerable number of mouse mutants die during embryonic development thereby hindering the analysis and interpretation of developmental phenotypes. The organ of Corti from mutant mice that die before birth can be cultured so that their in vitro development and responses to different factors can be analyzed. Additionally, we describe a technique for electroporating embryonic cochlear explants ex vivo which can be used to downregulate or overexpress specific gene(s) and analyze their potential endogenous function and test whether specific gene product is necessary or sufficient in a given context to influence mammalian cochlear development1-8.

We present a method that describes isolation and culture of cochlear explants from embryonic mouse inner ear. We also demonstrate a method for gene transfer into cochlear explants via square-wave electroporation. The in vitro explant culture coupled with gene transfer technique enables researchers to study the effects of altering gene expression during development.

Electroporation에 의한 배아 마우스 달팽이의 Explants의 문화 유전자 전송

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair ...

연구

발생학

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during oogenesis. In order to study roles of such maternal factors in early embryonic development, it is desirable to manipulate their functions from the very beginning of embryonic development. Conventional ways of gene interference are achieved by injection of antisense oligonucleotides (oligos) or mRNA into fertilized eggs, enabling under- or over-expression of specific proteins, respectively. However, these methods normally require more than several hours until protein expression is affected, and, hence, the interference of gene functions is not effective during early embryonic stages. Here, we introduce an experimental system in which expression levels of maternal proteins can be altered before fertilization. Xenopus laevis oocytes obtained from ovaries are defolliculated by incubating with enzymes. Antisense oligos or mRNAs are injected into defolliculated oocytes at the germinal vesicle (GV) stage. These oocytes are in vitro matured to eggs at the metaphase II (MII) stage, followed by intracytoplasmic sperm injection (ICSI). By this way, up to 10% of ICSI embryos can reach the swimming tadpole stage, thus allowing functional tests of specific gene knockdown or overexpression. This approach can be a useful way to study roles of maternally stored factors in early embryonic development.

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

We describe methods of manipulating Xenopus laevis immature oocytes, in vitro maturation of oocytes to eggs, and intracytoplasmic sperm injection. This protocol allows degradation of some maternal proteins and overexpression of genes of interest at fertilization, and hence is valuable to study roles of specific factors in early embryonic development.

조작 및<em&gt; 체외</em의&gt; 성숙<em&gt; 아프리카 발톱 개구리 laevis의</em세포질 내 정자 주입에 이어&gt; 난 모세포, 배아 발달을 연구하는

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during ...

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo development due to their similar developmental process. Although genetic factors are known to affect early embryonic development, the discovery of such factors has been a serious challenge. Microarray technology allows quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. One of the main decisions that have to be made when planning a microarray experiment is whether to use a one- or two-color approach. Two-color design increases technical replication, minimizes variability, improves sensitivity and accuracy as well as allows having loop designs, defining the common reference samples. Although microarray is a powerful biological tool, there are potential pitfalls that can attenuate its power. Hence, in this technical paper we demonstrate an optimized protocol for RNA extraction, amplification, labeling, hybridization of the labeled amplified RNA to the array, array scanning and data analysis using the two-color analysis strategy.

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Microarray technology allows quantitative measurement and gene expression profiling of transcripts on a genome-wide basis. Therefore, this protocol provides an optimized technical procedure in a two-color custom made bovine array using Day 7 bovine embryos to demonstrate the feasibility of using low amount of total RNA.

의 사체 프로파일 링<em&gt; 생체</em2 색의 마이크로 어레이 플랫폼을 사용하여&gt; 제작 소 착상 전 배아

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo ...

Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated by transcription factors which bind genomic regulatory regions including enhancers and promoters of cardiac constitutive genes. DNA is wrapped around histones that are subjected to chemical modifications. Modifications of histones further lead to repressed, activated or poised gene transcription, thus bringing another level of fine tuning regulation of gene transcription. Embryonic Stem cells (ES cells) recapitulate within embryoid bodies (i.e., cell aggregates) or in 2D culture the early steps of cardiac development. They provide in principle enough material for chromatin immunoprecipitation (ChIP), a technology broadly used to identify gene regulatory regions. Furthermore, human ES cells represent a human cell model of cardiogenesis. At later stages of development, mouse embryonic tissues allow for investigating specific epigenetic landscapes required for determination of cell identity. Herein, we describe protocols of ChIP, sequential ChIP followed by PCR or ChIP-sequencing using ES cells, embryoid bodies and cardiac specific embryonic regions. These protocols allow to investigating the epigenetic regulation of cardiac gene transcription.

A fine tuning regulation of gene transcription underlies embryonic cell fate decision. Herein, we describe chromatin immunoprecipitation assays used to investigate epigenetic regulation of both cardiac differentiation of stem cells and cardiac development of mouse embryos.

배아 줄기 세포와 조직의 심장 차별화의 성적인 규정

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated ...

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 ...

Culture of Murine Embryonic Metatarsals: A Physiological Model of Endochondral Ossification

The fundamental process of endochondral ossification is under tight regulation in the healthy individual so as to prevent disturbed development and/or longitudinal bone growth. As such, it is imperative that we further our understanding of the underpinning molecular mechanisms involved in such disorders so as to provide advances towards human and animal patient benefit. The mouse metatarsal organ explant culture is a highly physiological ex vivo model for studying endochondral ossification and bone growth as the growth rate of the bones in culture mimic that observed in vivo. Uniquely, the metatarsal organ culture allows the examination of chondrocytes in different phases of chondrogenesis and maintains cell-cell and cell-matrix interactions, therefore providing conditions closer to the in vivo situation than cells in monolayer or 3D culture. This protocol describes in detail the intricate dissection of embryonic metatarsals from the hind limb of E15 murine embryos and the subsequent analyses that can be performed in order to examine endochondral ossification and longitudinal bone growth.

We present a protocol to dissect and culture embryonic day 15 (E15) murine metatarsal bones. This highly physiological ex vivo model of endochondral ossification provides conditions closer to the in vivo situation than cells in monolayer or 3D culture and is a vital tool for investigating bone growth and development.

쥐 배아 중족골의 문화 : 연골 골화의 생리 학적 모델

The fundamental process of endochondral ossification is under tight regulation in the healthy individual so as to prevent disturbed development and/or ...

Dissection and Culture of Mouse Embryonic Kidney

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney development, the two progenitor tissues, the ureteric bud and the metanephric mesenchyme, communicate and reciprocally induce cellular mechanisms to eventually form the collecting system and the nephrons of the kidney. As mammalian embryos grow intrauterine and therefore are inaccessible to the observer, an organ culture has been developed. With this method, it is possible to study epithelial-mesenchymal interactions and cellular behavior during kidney organogenesis. Furthermore, the origin of congenital kidney and urogenital tract malformations can be investigated. After careful dissection, the metanephric rudiments are transferred onto a filter that floats on culture medium and can be kept in a cell culture incubator for several days. However, one must be aware that the conditions are artificial and could influence the metabolism in the tissue. Also, the penetration of test substances could be limited due to the extracellular matrix and basal membrane present in the explant.
One main advantage of organ culture is that the experimenter can gain direct access to the organ. This technology is cheap, simple, and allows a large number of modifications, such as the addition of biologically active substances, the study of genetic variants, and the application of advanced imaging techniques.

This protocol describes a method for isolating and culturing metanephric rudiments from mouse embryos.

마우스 배아 신장의 해부 및 배양

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

꼬마 선 충 생식 핵의 분포, 단백질, 및 골격을 전산 분석

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Isolation of Embryonic Tissues and Formation of Quail-Chicken Chimeric Organs Using The Thymus Example

The capacity to isolate embryonic tissues was an essential step for establishing the quail-chicken chimera system, which in turn has provided undisputed contributions to unveiling key processes in developmental biology.
Herein is described an optimized method to isolate embryonic tissues from quail and chickens by microsurgery and enzymatic digestion while preserving its biological properties. After isolation, tissues from both species are associated in an in vitro organotypic assay for 48 h. Quail and chicken tissues can be discriminated by distinct nuclear features and molecular markers allowing the study of the cellular cross-talk between heterospecific association of tissues. This approach is, therefore, a useful tool for studying complex tissue interactions in developmental processes with highly dynamic spatial modifications, such as those occurring during pharyngeal morphogenesis and the formation of the foregut endoderm-derived organs. This experimental approach was first developed to study the epithelial-mesenchymal interactions during early-stages of thymus formation. In this, the endoderm-derived prospective thymic rudiment and mesoderm-derived mesenchyme, were isolated from quail and chicken embryos, respectively.
The capacity of the associated tissues to generate organs can be further tested by grafting them onto the chorioallantoic membrane (CAM) of a chicken embryo. The CAM provides nutrients and allows gas exchanges to the explanted tissues. After 10 days of in ovo development, the chimeric organs can be analyzed in the harvested explants by conventional morphological methods. This procedure also allows studying tissue-specific contributions during organ formation, from its initial development (in vitro development) to the final stages of organogenesis (in ovo development).
Finally, the improved isolation method also provides three-dimensionally (3D) preserved embryonic tissues, that can also be used for high-resolution topographical analysis of tissue-specific gene-expression patterns.

This article provides a method to isolate pure embryonic tissues from quail and chicken embryos that can be combined to form ex vivo chimeric organs.

배아 조직과 Thymus 예제를 사용 하는 메 추 라 기-닭고기 공상 기관의 형성의 절연

The capacity to isolate embryonic tissues was an essential step for establishing the quail-chicken chimera system, which in turn has provided undisputed ...

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One challenge is that embryogenesis dynamically unfolds over a period of about 13 h; this half day-long timescale has constrained the scope of experiments by limiting the number of embryos that can be imaged. Here, we describe a semi-high-throughput protocol that allows for the simultaneous 3D time-lapse imaging of development in 80&#8211;100 embryos at moderate time resolution, from up to 14 different conditions, in a single overnight run. The protocol is straightforward and can be implemented by any laboratory with access to a microscope with point visiting capacity. The utility of this protocol is demonstrated by using it to image two custom-built strains expressing fluorescent markers optimized to visualize key aspects of germ-layer specification and morphogenesis. To analyze the data, a custom program that crops individual embryos out of a broader field of view in all channels, z-steps, and timepoints and saves the sequences for each embryo into a separate tiff stack was built. The program, which includes a user-friendly graphical user interface (GUI), streamlines data processing by isolating, pre-processing, and uniformly orienting individual embryos in preparation for visualization or automated analysis. Also supplied is an ImageJ macro that compiles individual embryo data into a multi-panel file that displays maximum intensity fluorescence projection and brightfield images for each embryo at each time point. The protocols and tools described herein were validated by using them to characterize embryonic development following knock-down of 40 previously described developmental genes; this analysis visualized previously annotated developmental phenotypes and revealed new ones. In summary, this work details a semi-high-throughput imaging method coupled with a cropping program and ImageJ visualization tool that, when combined with strains expressing informative fluorescent markers, greatly accelerates experiments to analyze embryonic development.

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

This work describes a semi-high-throughput protocol that allows simultaneous 3D time-lapse imaging of embryogenesis in 80&#8211;100 C. elegans embryos in a single overnight run. Additionally, image processing and visualization tools are included to streamline data analysis. The combination of these methods with custom reporter strains enables detailed monitoring of embryogenesis.