이 연구는 [부분적으로] 보건원, 국립 연구소의 환경 건강 과학의 교내 연구 프로그램에 의해 지원 되었다. 통계 분석에 대 한 특별 Shyamal Peddada 감사 합니다. 우리는 또한 험프리 야 오, 게리 조류, 유키 아 라오 그의 지속적인 지원과 조언을 위해이 원고, 및 프랑코 DeMayo의 검토를 위해 감사 하고자.

모든 작업은 마우스 코어의 NIEHS 노크 밖으로 인해 시설 정상 작동의 일환으로 수행 되었다. 모든 마우스는 12시 12분에 유지 했다 h 빛: 다크 사이클, NIH-31와 물 다이어트를 공급 했다 ad libitum. 모든 실험 동물 관리 및 사용 위원회는 국립 연구소의 환경 건강 과학의 수행 했다.
1. 동물 준비
<ol><li>기증자 쥐, b 6 번 식 (Cg)-Tyr < c-2J > b 6 자연스럽 게 나이의 8-10 주에 /J 여성 쥐 (Cg)-Tyr < c-2J > /J 남성 쥐 나이의 8과 26 주 사이. 시각적으로 플러그 다음 아침 (E0.5)을 확인 합니다.</li>
	<li>받는 사람 마우스, 스위스 웹스터 여성 쥐 나이의 6 그리고 9 주 사이 자연스럽 게 vasectomized 스위스 웹스터 남성 쥐 사육 번 식. 시각적으로 플러그 다음 아침 (E0.5)을 확인 합니다.</li>
</ol>2. ES 세포 준비
<ol><li>129-derieved AB2.2 ES 세포에 대 한 문화 15 보충 DMEM 셀 %FBS, 1% 글루타민, 1% 펜/strep, 및 0.1% β-mercaptoethanol. 그들의 권장된 프로토콜을 사용 하 여 ES 셀 공급 업체의 지시 당 다른 모든 ES 세포 문화.</li>
</ol>3. 배아 컬렉션
<ol><li>
동물 해 부
	<ol>
<li>여러 가지 60mm 요리, 약 1 5 마우스 플러스 10 mL 폭발 컬렉션 미디어 (BCM)와 3 추가 준비 (10 %FBS DMEM), 그리고 한 4 잘 잘 당 0.5 mL BCM으로 접시. 37 ° C, 5% CO2에서 equilibrate.</li>
		<li>B 6를 안락사 (Cg)-Tyr < c-2J > 10%의 유량과 CO2 질를 사용 하 여 E3.5에 /J 여성 쥐.</li>
		<li>Euthanizing, 후 부정사 위치에 쥐를 놓고 젖은 70% 에탄올으로 깨끗이 복 부.</li>
		<li>가능한 넓은 개방을 만드는 복 부의 중앙에만, 피부를 통해 첫 번째 절 개를 확인 합니다. 다음 다시 가능한 광범위 한 절 개를 만드는 복 벽을 통해 열. 참고:이 두 단계에 일을 줄이거나 모피 오염 제거에 도움이 됩니다.</li>
		<li>난소에서 시작 하 고 무딘 집게와 난소의 지방 패드를 들고 조심 스럽게 난소, 수 란 관, 및 지방 조직을 최소로 유지 하 여분 배려를 복용 하는 자 궁을 제거. 하나의 단일 단위로 또는 다음 절차에서 변경 되지 않고 개별 뿔으로 자 궁을 제거 합니다.</li>
		<li>제거 후 자 궁 폭발 컬렉션 미디어를 포함 하는 60 m m 접시에 놓습니다.</li>
	</ol>
</li>
	<li>
배아 제거
	<ol>
<li>BCM, 가득 10 mL 주사기를 준비 하 고 25g 바늘으로 적합.</li>
		<li>한 번에 한 자 궁 BCM의 지주 접시에서 그들을 제거, 과잉 혈액을 제거 하 고 해 부 현미경 저 배율에서 60 mm 접시에 배치 하는 조직에서 그들을 얼룩.</li>
		<li>신중 하 고 부드럽게 집게 해 부와 (수 란 관) 근처 선단부 바로 아래 자 궁을 잡고, 병렬 가능한 비행기로에 자 궁의 중앙에 바늘을 삽입 합니다. 그냥 넘어는 집게의 끝에 바늘을 삽입 합니다.</li>
		<li>바늘, 자 궁을 겸 자에 압력을 적용 하 고 약 1 mL의 자 궁 경적을 통해 BCM의 추방. 참고: 합니다 주의이 단계에서 주사기에 너무 많은 압력, 미디어의 통제 스트림 하면로 자주 요리를 떠나.</li>
		<li>60 mm 접시 당 최대 5 전체 uteri 자 궁 홍 후 컬렉션까지 인큐베이터에 접시를 놓습니다.</li>
	</ol>
</li>
	<li>
배아 컬렉션
	<ol>
<li>손으로 그린된 유리 피 펫 및 입 피 펫 장치 using, blastocysts 25 X 35 X 사이의 설정 해 범위에서 수집 합니다. (그림 1)입니다. 참고: 유연한 튜브의 30-40 cm의 입 피 펫 장치 구성, 5mm 미만 아이디 삽입 1, 000 µ L 피 펫 팁, 처음에 한쪽 끝을 가리키고 접사에 유리 피 펫. 연결 1 cc 주사기 배럴, 아니 플런저, 입 조각으로, 다른 끝에 주사기 필터를 추가 합니다.</li>
		<li>인큐베이터에서 요리를 제거 하 고 격자 패턴을 사용 하 여 태아에 대 한 접시의 모든 부분을 검사 합니다. 배아 선택은 개별적으로 하 고 보조 접시에서 BCM의 드롭에.</li>
		<li>모든 요리에서 수집 후 신선한 BCM, 한 방울에 배아를 한 번 더 세척 하 고 주입을 기다리고 4 잘 접시에 놓습니다.</li>
	</ol>
</li>
</ol>4. 태아 주입
<ol><li>확인에 따라 주사 바늘으로 개별적으로 ES 세포를 선택 합니다.<ol>
<li>5-7 mL 주입 미디어를 추가 하 여 주입 접시를 준비 (DMEM, 10 %FBS 및 20 mM HEPES) 유리 하단 주입 접시. 주사는 상 온에서 수행 됩니다.</li>
		<li>공기 또는 기름 microinjector를 사용 하 여 ES 세포를 주사 바늘을 진공을 적용 됩니다. 셀 사이의 가능한 작은 공간으로 유지 하는 것을 주의. 여기, 또는 사출 단계에서 ES 세포를 계산.</li>
		<li>ES 셀을 셀, 매끄러운 표면, 그리고 명백한 결함이 그들 등의 일반 인구에 비해 크기가 작은 매체를 선택 합니다. 참고: ES 세포 인구 형태학 품질에 크게 변화 하 고 사용 되는 표준 그들과 함께 조정 해야 합니다.</li>
	</ol>
</li>
	<li>약 15 ES 세포 (그림 1)와 배아를 주사.<ol>
<li>연결 지주 피 펫 팁 두 번째 공기 microinjector를 및 소유자의 오프닝 근처 태아 위치. 소유자에 게 태아를 보호 하는 진공을 적용 합니다.</li>
		<li>태아는 구역에 잘 집중 하 여 다음 슬라이드를 만지고 그냥은 그래야 지주 피펫으로 위나 아래로 이동 하 여 z 축에 대 한 조정. ES 세포 끝 근처 초점이 되도록 다음, 주사 바늘의 Z 축을 조정 합니다.</li>
		<li>부드럽게 주사 바늘 위쪽 이나 아래쪽으로 원하는 위치에 ICM을 넣어 회전에서 추진 하 여 지금 배아를 조정 합니다.</li>
		<li>구역, 그리고 ICM 또는 확고 하지만 제어 푸시를 사용 하 여 trophoblast 통해 주사 바늘을 삽입 합니다. 붕괴에서 blastocoel를 유지 하기 위해 주사 바늘의 microinjector에 약간의 압력을 적용 됩니다.</li>
		<li>바늘은 blastocoel를 침투 하 고, 일단 태아의 blastocoel ES 세포를 전송 하는 microinjector에서 압력을 적용. 바늘의 경사로 잠시 일시 중지 셀 유지 하면서 정상으로 돌아갑니다 배아에 압력을 허용 하는 배아를 종료 하기 시작 합니다. 참고: 참조 그룹 ES 세포는 일반적으로 주입 했다, 전통에 따라 세포 주입 프로토콜9, ICM을 터치 하지 않도록 주의 복용. ES 세포 또한 ICM, 직접 통해는 ICM (TICM) (그림 2보충 영화) 바늘을 밀어를 통해 주입 했다.</li>
	</ol>
</li>
</ol>5입니다. eEmbryos의 전송
<ol><li>제조의 절차 당 비 외과 배아 전송 장치를 사용 하 여 E2.5에서 스위스 웹스터 마우스 배아를 전송 합니다. 이 아닌-수술 마 취 또는 수술에 특별 한 주의 요구 한다. 집 쥐의 배아, 그리고 배달 및 후속 이유를 통해 전송 후 개별적으로.</li>
</ol>

<ol>
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저자는 공개 없습니다 경쟁 관심사 있다.

그것은 오래는 ICM의 어떤 소란 이어질 수 사망, 사실, 현재 blastocyst microinjection 프로토콜 여전히이12,13의 경고 생각 되었습니다. 그것은 우리가 여기에 표시는 ICM 통해 microinjection는 태아에 게 해로운 고 키메라의 수확량을 증가.
이 효과 대 한 정확한 메커니즘 확인 하지 했다. 그러나, ICM와 함께 hypoblast의 기계적 장애 ES 세포 통합 mic...

거의 25 년 동안, 유전자 표적으로 쥐의 창조 자주 키메라를 전송 하는 생식의 세대를 위한 blastocysts ES 세포 microinjection 단계에서 병목 현상에 의해 방해, 느린 과정 되었습니다. CRISPR/Cas91,2 ES 세포 및 높은 처리량 ES에 타겟팅을 포함 한 최근 전진 호 같은 세대 컨소시엄 세포, 유전자 변형된 ES 세포의 생성/가용성 향상. 그러나, 생식 키메라의 생성이 ES 세포3,4에서 유전자 변형된 쥐를 만들기에 병목을 남아 있다. 높은 처리량 ES 세포 생성 프로젝트 생식 키메라의 성공적인 창조에 대 한 중요 한 ES 세포 품질 높은 가변성에 의해 괴 롭 혀 왔다. 또한, 일반적으로 이용한 ES 세포 선의 일부 높은 이수성, 및 생식 유능한 키메라5의 어려운 생산 알려져 있다.
어느 높은 키메라와 쥐를 만들기 위한 많은 방법은 개발 되었다 또는 완전히 ES 세포 파생 마우스6,7,8,,910. 이러한 시스템의 각 그것의 자신의 공로 있다, 그러나 그들은 또한 그들의 결점 있다. Tetraploid chimeras, 마우스, 파생 된 100 %ES 세포를 생성 하는 동안 생성, 비효율적 이며 outbred 라인 5,6에 일반적으로 제한 된. Morula 주입의 새로운 방법 높은 백분율 chimeras, 100%에 접근을 얻을 수 있지만 일반적으로 상당한 추가 장비 (레이저 또는 piezos) 및 훈련10,11필요로 합니다. 이러한 기술은 이미 자리에는 실험실,이 새로운 방법론 필요 없을 수 있습니다.
이 연구의이 목적은 키메라 세대, 이후 마우스 식민지를 설치 하는 돌연변이 체 대립 유전자의 생식 전송의 기회를 증가의 속도 높이기 위해 일반적인 기술과 쉽게 사용할 수 있는 장비를 사용 하는 메서드를 찾아야 했다.

<table><tbody><tr><td>ES Cell injection needle</td><td>Humagen</td><td>MSC-20-0</td><td></td></tr><tr><td>NSET device</td><td>Paratechs</td><td>60010</td><td></td></tr><tr><td>DMEM</td><td>Gibco (life technologies)</td><td>11965-092</td><td></td></tr><tr><td>FBS</td><td>Gibco (life technologies)</td><td>10439-024</td><td>lots should be individually tested for ES Cell compatibility.</td></tr><tr><td>HEPES</td><td>Sigma</td><td>H0887</td><td></td></tr><tr><td>&amp;#946;-mercaptoethanol</td><td>Sigma</td><td>M7522</td><td></td></tr><tr><td>Micro manipulator</td><td>Leica</td><td>Micro manipulator</td><td></td></tr><tr><td>micro injector</td><td>Eppendorf AG</td><td>Cell Tram Air 5176</td><td></td></tr><tr><td>Microscope</td><td>Leica</td><td>DM IRB</td><td></td></tr><tr><td>4 well dish</td><td>Thermo Scientific</td><td>176740</td><td></td></tr><tr><td>60 mm dish</td><td>Sarstedt</td><td>83.3901</td><td></td></tr><tr><td>B6(Cg)-Tyr&amp;#60;c-2J&amp;#62;/J</td><td>Jackson Labs, Bar Harbor, Maine, USA</td><td>000058</td><td></td></tr><tr><td>Swiss Webster mice</td><td>Taconic, USA</td><td>Tac:SW</td><td></td></tr><tr><td>Injection Dish</td><td>MatTek Corp.</td><td>P50G-0-30-F</td><td></td></tr></tbody></table>

trans-inner cell mass injection of embryonic stem cells leads to higher chimerism rates

ES 셀 방법론을 통해 유전자 변형된 생쥐의 창조에 효율성을 증가 하는 노력에서 선물이 현재 blastocyst 주입 프로토콜을 적응. 여기 우리 보고 배아, 그리고 트랜스 안 세포 질량 (TICM)를 통해 주입의 간단한 회전 50%, 추가 장비에 31%에서 공상 쥐의 비율을 증가 또는 추가 훈련을 전문. 26 다른 근 친 클론, 그리고 9 개월의 기간 동안 35 총 클론 주입 했다. 전통적인 주입 기술 및 TICM 사이 임신 속도 또는 배아의 회복 속도에 상당한 차이가 있었습니다. 따라서 주입 프로세스와는 blastocyst의 간단한 위치는 ICM을 통해 주입에 모든 주요 변경 없이 blastocoel 구멍에 ES 세포 방출 잠재적으로 향상 시킬 수 있습니다 공상 생산 이후 수량 생식 전송입니다.

임의 효과로 셀-선 치료, 혼합된 효과 선형 모델 소프트웨어 (예: SAS (버전 9.2))에서 데이터를 분석 하 사용 되었다. 각 분석 배아 전송 및 셀 라인의 수에 대 한 제어. 두 번째 분석 또한 살아남은 새끼의 수에 대 한 제어.
이 연구를 위해 각 ES 세포 선 및 복제 주입 했다 전통 둘 다 및 TICM 기법, 각 배아 후 두 가지 방법 사이의 교?...

Watch this Scientific Journal Video about Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates at JoVE.com

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

여기 우리는 새로운 장비를 사용 하지 않고 키메라 생산을 증가 하는 프로토콜을 제시. 주입에 대 한 배아의 단순한 방향 변경 생산, 배아의 수를 증가 하 고 잠재적으로 생식 전송 타임을 줄일 수 있습니다.

트랜스-내부 세포 배아 줄기 세포의 대량 주입 리드 더 높은 Chimerism 요금

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

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Research

JoVE Journal

Biology

10.2K 조회수.  NIEHS/NIH. 여기 우리는 새로운 장비를 사용 하지 않고 키메라 생산을 증가 하는 프로토콜을 제시. 주입에 대 한 배아의 단순한 방향 변경 생산, 배아의 수를 증가 하 고 잠재적으로 생식 전송 타임을 줄일 수 있습니다.

비디오: Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged antigen-specific T cell receptor (TCR) have been an indispensable tool to study T cell development and function. However, such TCRs are usually isolated from the relevant T cells after long-term culture often following repeated antigen stimulation, which unavoidably selects for T cells with high affinity. Random genomic integration of the TCR &alpha;- and &beta;-chain and expression from non-endogenous promoters can lead to variations in expression level and kinetics.
Epigenetic reprogramming via somatic cell nuclear transfer provides a tool to generate embryonic stem cells and mice from any cell of interest. Consequently, when SCNT is applied to T cells of known specificity, these genetic V(D)J rearrangements are transferred to the SCNT-embryonic stem cells (ESCs) and the mice derived from them, while epigenetic marks are reset. We have demonstrated that T cells with pre-defined specificities against Toxoplasma gondii can be used to generate mouse models that express the specific TCR from their endogenous loci, without experimentally introduced genetic modification. The relative ease and speed with which such transnuclear models can be obtained holds promise for the construction of other disease models.

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

We demonstrate here that epigenetic reprogramming via Somatic Cell Nuclear Transfer (SCNT) can be used as a tool to generate mouse models with pre-defined T cell receptor (TCR) specificities. These transnuclear mice express the corresponding TCR from their endogenous locus under the control of the endogenous promoter.

반대 미리 정의된 T 세포 수용체 Specificities와 Transnuclear 쥐 Toxoplasma gondii SCNT 비아 획득

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged ...

연구

면역학 및 감염병학

Methods for Precisely Localized Transfer of Cells or DNA into Early Postimplantation Mouse Embryos

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, cell culture has been widely used in developmental biology studies. However, it is important to determine whether in vitro cultured cells truly represent in vivo cell types. Grafting cells into embryos, followed by an assessment of their contribution during development is a useful method to determine the potential of in vitro cultured cells. In this study, we describe a method for grafting cells into a defined site of early postimplantation mouse embryos, followed by ex vivo culture. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing precise localization and adjustment of the number of cells receiving exogenous DNA with both high transfection efficiency and low cell death. These techniques, which do not require any specialized equipment, render experimental manipulations of the gastrulation and early organogenesis-stage mouse embryo possible, allowing analysis of commitment in cultured cell subpopulations and the effect of genetic manipulations in situ on cell differentiation.

We demonstrate a method for grafting cultured cells into defined sites of early mouse embryos to determine their in vivo potential. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing the precise delivery of exogenous DNA into a few cells in the embryos.

초기 Postimplantation 마우스 배아로 세포 나 DNA를 정확하게 현지화 전송하는 방법

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, ...

발생학

Combinational Treatment of Trichostatin A and Vitamin C Improves the Efficiency of Cloning Mice by Somatic Cell Nuclear Transfer

Somatic cell nuclear transfer (SCNT) provides a unique opportunity to directly produce a cloned animal from a donor cell, and it requires the use of skillful techniques. Additionally, the efficiencies of cloning have remained low since the successful production of cloned animals, especially mice. There have been many attempts to improve the cloning efficiency, and trichostatin A (TSA), a histone deacetylase inhibitor, has been widely used to enhance the efficiency of cloning. Here, we report a dramatically improved cloning method in mice. This somatic cell nuclear transfer method involves usage of Hemagglutinating virus of Japan Envelope (HVJ-E), which enables easy manipulation. Moreover, the treatment using two small molecules, TSA and vitamin C (VC), with deionized bovine serum albumin (dBSA), is highly effective for embryonic development. This approach requires neither additional injection nor genetic manipulation, and thus presents a simple, suitable method for practical use. This method could become a technically feasible approach for researchers to produce genetically modified animals from cultured cells. Furthermore, it might be a useful way for the rescue of endangered animals via cloning.

We describe a dramatically improved method for mouse cloning using trichostatin A, vitamin C, and deionized bovine serum albumin. We show a simplified, reproducible protocol that supports efficient development of cloned embryos. Hence, this method could become a standardized procedure for mouse cloning.

Trichostatin A와 비타민 C의 조합 치료 체세포 핵 이식에 의해 생쥐 복제의 효율성 향상

Somatic cell nuclear transfer (SCNT) provides a unique opportunity to directly produce a cloned animal from a donor cell, and it requires the use of ...

Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups of cells in a larger context are altered.  Watching how one cell interacts with and behaves in a unique environment is essential to characterizing cell functions.  Determining how the localized misexpression of a specific protein influences surrounding cells provides insightful information on the roles that protein plays in a variety of developmental processes.  Our lab uses the zebrafish model system to uniquely combine genetic approaches with classical transplantation techniques to generate genotypic or phenotypic chimeras.  We study neuron-glial cell interactions during the formation of forebrain commissures in zebrafish. This video describes a method that allows our lab to investigate the role of astroglial populations in the diencephalon and the roles of specific guidance cues that influence projecting axons as they cross the midline. Due to their transparency zebrafish embryos are ideal models for this type of ectopic cell placement or localized gene misexpression. Tracking transplanted cells can be accomplished using a vital dye or a transgenic fish line expressing a fluorescent protein. We demonstrate here how to prepare donor embryos with a vital dye tracer for transplantation, as well as how to extract and transplant cells from one gastrula staged embryo to another.  We present data showing ectopic GFP+ transgenic cells within the forebrain of zebrafish embryos and characterize the location of these cells with respect to forebrain commissures.  In addition, we show laser scanning confocal timelapse microscopy of Alexa 594 labeled cells transplanted into a GFP+ transgenic host embryo.  These data provide evidence that gastrula staged transplantation enables the targeted positioning of ectopic cells to address a variety of questions in Developmental Biology.

Zebrafish cell transplantation enables the combination of genetics and embryology to generate tissue specific chimeras. This video demonstrates gastrula staged cell transplantations that have allowed our lab to investigate the roles of astroglial populations and specific guidance cues during commissure formation in the forebrain.

조직 타겟 배아 Chimeras : Zebrafish Gastrula 세포 이식

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups ...

생물학

Intravenous and Intra-amniotic In Utero Transplantation in the Murine Model

In utero transplantation (IUT) is a unique and versatile mode of therapy that can be used to introduce stem cells, viral vectors, or any other substances early in the gestation. The rationale behind IUT for therapeutic purposes is based on the small size of the fetus, the fetal immunologic immaturity, the accessibility and proliferative nature of the fetal stem or progenitor cells, and the potential to treat a disease or the onset of symptoms prior to birth. Taking advantage of these normal developmental properties of the fetus, the delivery of hematopoietic stem cells (HSC) via an IUT has the potential to treat congenital hematologic disorders such as sickle cell disease, without the required myeloablative or immunosuppressive conditioning required for postnatal HSC transplants. Similarly, the accessibility of progenitor cells in multiple organs during development potentially allows for a more efficient targeting of stem/progenitor cells following an IUT of viral vectors for gene therapy or genome editing. Additionally, IUT can be used to study normal developmental processes including, but not limited to, the development of immunologic tolerance. The murine model provides a valuable and affordable means to understanding the potential and limitations of IUT prior to pre-clinical large animal studies and an eventual clinical application. Here, we describe a protocol for performing an IUT in the murine fetus through intravenous and intra-amniotic routes. This protocol has been used successfully to elucidate the necessary conditions and mechanisms behind in utero hematopoietic stem cell transplantation, tolerance induction, and in utero gene therapy.

Intravenous and Intra-amniotic In Utero Transplantation in the Murine Model

We describe a protocol for performing an in utero transplantation (IUT) through intravenous and intra-amniotic routes of injection in the murine model. This protocol can be used to introduce cells, viral vectors, and other substances into the unique immune-tolerant fetal environment.

정 맥 및 양수 내 Utero Murine 모델에 이식

In utero transplantation (IUT) is a unique and versatile mode of therapy that can be used to introduce stem cells, viral vectors, or any other substances ...

Microinjection-based System for In Vivo Implantation of Embryonic Cardiomyocytes in the Avian Embryo

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a general challenge in developmental biology research. In the embryonic heart, this can be particularly difficult as regional differences in the expression of transcriptional regulators, paracrine/juxtacrine signaling cues, and hemodynamic force are all known to influence cardiomyocyte maturation. A simplified method to alter a developing cardiomyocyte's molecular and biomechanical microenvironment would, therefore, serve as a powerful technique to examine how local conditions influence cell fate and function. To address this, we have optimized a method to physically transplant juvenile cardiomyocytes into ectopic locations in the heart or the surrounding embryonic tissue. This allows us to examine how microenvironmental conditions influence cardiomyocyte fate transitions at single cell resolution within the intact embryo. Here, we describe a protocol in which embryonic myocytes can be isolated from a variety of cardiac sub-domains, dissociated, fluorescently labeled, and microinjected into host embryos with high precision. Cells can then be directly analyzed in situ using a variety of imaging and histological techniques. This protocol is a powerful alternative to traditional grafting experiments that can be prohibitively difficult in a moving tissue such as the heart. The general outline of this method can also be adapted to a variety of donor tissues and host environments, and its ease of use, low cost, and speed make it a potentially useful application for a variety of developmental studies.

In this method, embryonic cardiac tissues are surgically microdissected, dissociated, fluorescently labeled, and implanted into host embryonic tissues. This provides a platform for studying the individual or tissue level developmental organization under ectopic hemodynamic conditions, and/or altered paracrine/juxtacrine environments.

조류 배아 배아 Cardiomyocytes의 생체 내 주입에 대 한 microinjection 기반 시스템

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a ...

Dechorionation of Medaka Embryos and Cell Transplantation for the Generation of Chimeras

Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the three vertebrate model organisms in which genome-wide phenotype-driven mutant screens were carried out 1. Divergence of functional overlap of related genes between medaka and zebrafish allows identification of novel phenotypes that are unidentifiable in a single species 2, thus medaka and zebrafish are complementary for genetic dissection of the vertebrate genome functions. Manipulation of medaka embryos, such as dechorionation, mounting embryos for imaging and cell transplantation, are key procedures to work on both medaka and zebrafish in a laboratory. Cell transplantation examines cell autonomy of medaka mutations. Chimeras are generated by transplanting labeled cells from donor embryos into unlabeled recipient embryos. Donor cells can be transplanted to specific areas of the recipient embryos based on the fate maps 3 so that clones from transplanted cells can be integrated in the tissue of interest during development. Due to the hard chorion and soft embryos, manipulation of medaka embryos is more involved than in zebrafish. In this video, we show detailed procedures to manipulate medaka embryos.

Due to the hard chorion and soft embryos, manipulation of medaka embryos is more involved than in zebrafish. This video shows step-by-step procedures for how to manipulate medaka embryos, including dechorionation, mounting in agarose for imaging and cell transplantation for the production of chimeras. These procedures are essential to use medaka and zebrafish in a laboratory to take full advantage of their complementary features for the genetic dissection of vertebrate genome functions.

Chimeras의 생성에 대한 Medaka의 태아 및 세포 이식의 Dechorionation

Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the three vertebrate model organisms in ...

Xenotransplantation of Human Stem Cells into the Chicken Embryo

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

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

치킨의 배아에 인간 줄기 세포의 Xenotransplantation

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

Single Cell Transfection in Chick Embryos

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a thorough analysis of the fates of single cells under normal and experimental conditions. To this end, transfection methods that target single progenitors are a prerequisite. We describe here a technically straightforward method for transfecting single cells in chicken tissues in-ovo, allowing reliable lineage tracing as well as genetic manipulation. Specific tissue domains are targeted within the somite or neural tube, and DNA is injected directly into the epithelium of interest, resulting in sporadic transfection of single cells. Using reporters, clonal populations may consequently be traced for up to three days, and behavior of genetically manipulated clonal populations can be compared with that of controls. This method takes advantage of the accessibility of the chick embryo along with emerging tools for genetic manipulation. We compare and discuss its advantages over the widely-used electroporation method, and possible applications and use in additional in-vivo models are also suggested. We advocate the use of this method as a significant addition and complement for existing lineage tracing and genetic interference tools.

Using fine tip micropipettes we inject plasmid DNA into subdomains of chicken somites or neural tubes. The concentration of the plasmid is adjusted to generate single transfected cells. We then allow the cells to develop into clonal populations.

칙 태아의 단일 세포 Transfection

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a ...

CRISPR/Cas9-Mediated Highly Efficient Gene Targeting in Embryonic Stem Cells for Developing Gene-Manipulated Mouse Models

The CRISPR/Cas9 system has made it possible to develop genetically modified mice by direct genome editing using fertilized zygotes. However, although the efficiency in developing gene-knockout mice by inducing small indel mutation would be sufficient enough, the efficiency of embryo genome editing for making large-size DNA knock-in (KI) is still low. Therefore, in contrast to the direct KI method in embryos, gene targeting using embryonic stem cells (ESCs) followed by embryo injection to develop chimera mice still has several advantages (e.g., high throughput targeting in vitro, multi-allele manipulation, and Cre and flox gene manipulation can be carried out in a short period). In addition, strains with difficult-to-handle embryos in vitro, such as BALB/c, can also be used for ESC targeting. This protocol describes the optimized method for large-size DNA (several kb) KI in ESCs by applying CRISPR/Cas9-mediated genome editing followed by chimera mice production to develop gene-manipulated mouse models.

Here we present a protocol for developing genetically modified mouse models using embryonic stem cells, especially for large DNA knock-in (KI). This protocol is tuned up using CRISPR/Cas9 genome editing, resulting in significantly improved KI efficiency compared with the conventional homologous recombination-mediated linearized DNA targeting method.

유전자 조작 마우스 모델 개발을 위한 배아 줄기 세포에서 CRISPR/Cas9 매개 고효율 유전자 표적화

The CRISPR/Cas9 system has made it possible to develop genetically modified mice by direct genome editing using fertilized zygotes. However, although the ...