우리는 읽고 원고를 편집 브래드 Lackford 감사드립니다. 이 연구는 환경 건강 과학, 건강 교내 연구 프로그램 Z01ES102745 국립 연구소 (GH까지)의 국립 연구소에 의해 지원되었다.

1. Oct4GiP 마우스 ES 세포 유지 보수 Oct4GiP 전지는 친절 박사 오스틴 스미스에 의해 제공되었다. 그들은 Oct4-GFPiresPac transgene 4,5 들고 129/Ola 마우스에서 파생되었다. 그들은 ESGRO 전체 더하기 clonal 학년 중간 (Millipore), 또는 M15 매체의 젤라틴 코팅 조직 배양 플레이트에 유지됩니다 DMEM (Invitrogen)은 15% FBS, 1000 U / ML ESGRO (Millipore), 1X 비로 보충 필수 아미노산 (Invitrogen), 1X EmbryoMax Nucleasides (Millipore) 및 10 μm의 β-메르 캅 토 에탄올. <ol><li> 30 분간 실온에서 0.1 % 젤라틴 (시그마) (0.1 ML 젤라틴 용액 / cm 2)와 코트 판. </li><li> 플레이트 Oct4GiP의 ~ 2시 젤라틴 코팅 접시에있는 세포 × 10 4 / cm 2. </li><li> 0.05 %의 트립신과 함께 이틀 세포를 분할. </li></ol> 2. Oct4GiP 셀에서 siRNA의 Transfection Oct4GiP 기자 분석은 대부분의 convenien입니다M15 매체 또는 접시 384도 (코닝 또는 BD) - tly 젤라틴 코팅 평평한 바닥에서 96 실시. 전체 절차는 그림 1에 설명되어있다. <ol><li> siRNA - 지질 단지 어셈블리 : <ol><li> 96 - 웰 플레이트에 transfection 들어, U-바닥에 siRNA - 지질 단지 96 - 웰 플레이트를 조립. </li><li> 각 우물로, 10 μl OptiMEM (Invitrogen)와 0.3 μl Lipofectamine 2000 (Invitrogen)를 혼합하고 5 분 동안 상온에서 품어. </li><li> 지질 OptiMEM 혼합물에 5 pmol siRNA를 추가하고 또 다른 15 분 동안 품어. siRNA : 6ul : 지질 비율이 100 pmol입니다. </li><li> 멀티 채널 피펫으로 젤라틴 코팅 평면 바닥 판으로 U-맨 아래 판에서 OptiMEM의 siRNA-지질 단지를 전송합니다. </li><li> 384 잘 접시에 transfection 들어, 0.1 μl Lipofectamine 2000 및 10 μl OptiMEM 2 pmol siRNA를 이용하여 siRNA - 지질 단지를 준비합니다. </li></ol></li></ol> 참고 : 최종 siRNA 농도의transfections 50 nm의입니다. 3-4 생물 학적을 수행하면 각 siRNA의 transfection을 위해 복제합니다. 플랫 바닥 젤라틴 코팅 플레이트로 U-바닥 플레이트와 나누어지는의 siRNA-지질 단지의 마스터 섞어 설정합니다. <ol start="2"><li> Oct4GiP 전지 도금 및 transfection : <ol><li> 96 - 웰 플레이트에 transfection 내용 Oct4GiP 세포를 수집 및 9 X 10 4 셀 / ML에 신선한 M15 매체에서 그들을 resuspend. </li><li> 잘 멀티 채널 피펫을 사용하여 각 세포 현탁액을 100 μl를 추가합니다. </li><li> 3-5 번 아래로 pipeting 및하여 우물에 사전 aliquoted siRNA-lipids의 단지와 세포 현탁액을 섞는다. 다른 siRNA의 transfections 대한 정보를 변경합니다. </li><li> 384 잘 번호판, resuspend의 Oct4GiP 세포의 Transfection을 위해 6 X 10 4 셀 / 각도에 대한 ML 및 나누어지는 30 μl 세포 현탁액. </li></ol></li></ol> 참고 : 최적의 셀 도금 밀도는 일반적으로 3 × 10 5 세포 / ㎝ 2입니다하지만 m불안하다는 크게 세포 성장이나 생존에 영향을 siRNAs에 대한 최적화가 필요합니다. 낮은 도금 밀도는 transfection 동안 가난한 세포 생존으로 이어질 것입니다. 높은 도금 밀도가 높은 세포 합류 유도 차별화에 의한 높은 배경으로 이어질 것입니다. 만약 2-4 × 10 5 세포 / ㎝ 2 사이의 필요한 테스트 도금 밀도. <ol start="3"><li> 매체 변화 : <ol><li> 다중 채널 흡인기 (코닝) 또는 진공 지팡이 (VP-학술)을 사용하여 오래된 매체를 제거합니다. 접시의 바닥에 전지를 긁히지 않게 조심해라. </li><li> 매일 멀티 채널 피펫을 사용하여 신선한 ES 세포 매체 (384 잘 접시 96 - 웰 플레이트에서 30 μl 100 μl)으로 세포를 먹이. </li></ol></li></ol> 3. FACS 분석 <ol><li> 세포 분열 : <ol><li> 나흘 transfection 후, 멀티 채널 흡인기 (코닝) 또는 진공 지팡이 (VP-학술)을 사용하여 매체를 제거합니다. </li><li> 96 - 웰 플레이트 들어, 100μ으로 한 세포를 헹굼리터 PBS. </li><li> 잘 멀티 채널 피펫을 사용하여 각 25 μl 0.25 %의 트립신을 추가합니다. </li><li> 가끔 교반과 오분 동안 상온에서 부화하고, 시각 세포의 완전한 분리를 보장하기 위해 검사한다. </li><li> 트립신을 inactivate 반복 pipetting하여 단일 세포 현탁액으로 세포를 떼어 놓다 10 % FBS와 PBS의 90 μl를 추가합니다. </li><li> 384 잘 접시의 경우 10 % FBS와 30 μl PBS 갖춘 10 μl 트립신과 끄지으로 세포를 끊을. </li></ol></li><li> FACS 분석 : <ol><li> HTS 유닛 또는 기타 유사 시설의 FACS 분석기 (예 : Accuri 또는 Intellicyt 등)을 갖춘 BD LSRII FACS 분석기에서 GFP의 형광 분석. </li><li> HTS의 높은 처리량 모드를 사용하여 세포 현탁액의 10 μl를 분석합니다. 1.0 X 10 4 세포로 카운트 임계값을 설정합니다. </li><li> 앞으로 대 사이드 분산형 음모에 세포를 캡처하는 PMT 전압과 임계값을 조정합니다. forw에서 라이브 세포 인구를위한 게이트ARD 대 사이드 분산형 계획. </li><li> GFP 채널에 대한 히스토그램 플롯을 만듭니다. 세포 ~ 10 %는 모방이나 제어 siRNA transfected 세포에 GFP-음수로 표시되도록 GFP 채널의 게이트를 설정합니다. </li><li> % 차별화된 세포 세포 = %의 GFP-음성 : 각 치료에서 % GFP-음성 세포를 결정합니다. 2 꼬리 T-테스트를​​ 사용하여 실험 및 제어 siRNA의 transfections 사이 % 차별화된 세포를 비교하고, P-값 &lt;0.01로 긍정적인 안타를 득점. </li></ol></li></ol> 4. 대표 결과 Oct4, Nanog, 그리고 Sox2는 ES 세포 자기 갱신 6,7,9-11의 유지에 중요한 역할을 세 유전자 있습니다. 그림 Oct4GiP 기자 분석이 쉽게 이러한 요인을 입을으로 인한 차별을 감지할 수있는이 공연 Oct4GiP ES 세포. 그림 2A는 ES로 유지하면 Oct4GiP의 ES 세포는 GFP-양수 보여줍니다<html> . 그림 2B 세포는 앞으로 대 측면 분산형 계획 및 제어 또는 Oct4-siRNAs로 transfected Oct4GiP 세포의 GFP 채널의 히스토그램을 보여줍니다. 죽은 세포와 파편 GFP-음성이며 배경을 증가하므로 그것은 앞으로 대 측면 분산형 플롯에서 라이브 세포에 대한 게이트가 필요합니다. 한편, 가능한 세포 대단히 짧은 시간을 제외하도록 차별을 이중어 항상 필요하지 않습니다. 제어 siRNA transfected 세포에서는 세포의 대부분은 GFP 양성해야합니다. 명백한 GFP - 부정적인 인구가 제어 siRNA transfected 우물에있는 경우, 시작 Oct4GiP 세포 또는 transfection 절차가 손상되었을 수 있으며 결과는 interpretable되지 않을 수 있습니다. 그림 2C는에서 % 차별화된 세포 (% 차별화된 세포 = % GFP-음성 세포)의 막대 그래프를 보여줍니다 제어, Oct4-, Nanog-, 그리고 Sox2-siRNA transfected 세포. <html> d/3987/3987fig1.jpg &quot;/&gt; 1 그림. Oct4GiP 기자 분석의 개요. <img alt="그림 2" src="/files/ftp_upload/3987/3987fig2.jpg" /> 그림 2. Oct4GiP 기자 분석은 Nanog, Oct4, Sox2 또는 입을 의한 ES 세포 분화를 검색할 수 있습니다. ) E14Tg2a (야생 형, 블랙 라인) 및 Oct4GiP (녹색 라인) 세포는 FACS로 분석되었다. GFP 채널의 히스토그램은 Oct4GiP 세포가 GFP 양성한지 확인하십시오. B) Oct4GiP 세포는 제어 또는 Oct4-siRNA와 96 - 웰 플레이트에 transfected와 FACS는 사일 transfection 후 분석했다 보여줍니다. 앞으로 transfected 세포의 GFP 채널의 측면 분산형 플롯과 histograms 수와 같습니다. C)가 Oct4GiP 전지로 transfected되었다 제어, Nanog -, Oct4-, 또는 Sox2-siRNA. % 차별화된 세포는 % 사일 transfection 후 세포가 GFP-음성으로 결정되었고, + / - 뜻으로 꾸몄다되었다 - 평균의 표준 오차 (N = 4).F = &quot;http://www.jove.com/files/ftp_upload/3987/3987fig2large.jpg&quot;대상 = &quot;_blank&quot;&gt; 큰 그림을 보려면 여기를 클릭하십시오.

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관심의 어떠한 충돌 선언 없습니다.

우리가 위에서 설명 Oct4GiP 기자 분석은 정량적 자기 갱신 대 분화의 정도를 측정할 수 있습니다. 이러한 형태학 기반의 12 확산 / assays 생존 기반과 같은 다른 가능한 방법, 비교, 그것은 높은 감도 및 처리량뿐만 아니라 ES 세포 상태의 더 직접적인 측정을 제공합니다. 그것은 따라서 잘 대형 스크린 및 유전자 epistasis 분석에 적합합니다. 실제로, 우리는 다른 사람이 성공적으로 게놈 전체의...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> ESGRO 완전한 플러스 clonal 등급 매체 </td><td> Millipore </td><td> SF001-500P </td></tr><tr><td> DMEM (높은 포도당 1X) </td><td> Invitrogen </td><td> 11,965 </td></tr><tr><td> 0.25 % 트립신-EDTA (에틸렌 다이아 민 테트라 초산) </td><td> Invitrogen </td><td> 25,200 </td></tr><tr><td> Lipofectamine 2000 </td><td> Invitrogen </td><td> 1,001,817 </td></tr><tr><td> OPTI-MEM (혈청 매체를 줄이기) </td><td> Invitrogen </td><td> 31,985 </td></tr><tr><td> ESGRO mLIF (10 7 units/1ml) </td><td> Millipore </td><td> DAM1776540 </td></tr><tr><td> MEM NEAA (비 필수 아미노산) </td><td> Invitrogen </td><td> 11,140 </td></tr><tr><td> ES 세포에 대한 100x Nucleosides </td><td> Millipore </td><td> 10620-1 </td&gt; </tr><tr><td> 2 - 메르 캅 토 에탄올 </td><td> 시그마 </td><td> M7522-100ml </td></tr><tr><td> ES-자격 태아 소 혈청 </td><td> Invitrogen </td><td> 10,437 </td></tr><tr><td> Nanog의 siRNA </td><td> Invitrogen </td><td> MSS231181 </td></tr><tr><td> Oct4 siRNA </td><td> Dharmacon </td><td> D-046256-02 </td></tr><tr><td> Sox2의 siRNA </td><td> Dharmacon </td><td> M-058489-01 </td></tr></tbody></table> 제어 siRNA : Dharamcon에 의해 합성 반딧불 루시페라제을 (5&#39;-CGTACGCGGAATACTTCGA) 대상으로 s​​iRNA의 이중.

oct4gip reporter assay to study genes that regulate mouse embryonic stem cell maintenance and self-renewal

Pluripotency과 자기 갱신은 배아 줄기 세포의 두 정의 특성 (ES 세포)입니다. 기본적인 분자 메커니즘을 이해하는 것은 크게 발달 생물학 연구, 질병 모델링, 약물 발견 및 재생 의료 (1,2 년 검토)의 ES 세포의 사용을 촉진합니다. ES 세포 유지 및 자기 갱신의 소설 규제의 식별 및 특성을 신속하게하기 위해 우리는 양적 Oct4GiP 세포에게 3을 사용 마우스 ES 세포의 자기 갱신 상태를 측정하는 형광 리포터 기반의 분석을 개발했습니다. Oct4GiP 전지는 4,5 Oct4 유전자 프로 모터 영역의 통제하에 녹색 형광 단백질 (GFP)을 표현. Oct4는 차별 6,7 중에 ES 세포 자기 갱신이 필요합니다, 그리고 고도의 ES 세포에 표현되며, 빠르게 아래쪽 규제. 그 결과, 기자 세포에서 GFP 발현과 형광 성실하게 상호ES 세포 정체성 5 및 형광 - 활성 셀 정렬과 (FACS) 분석 밀접하게 단세포 레벨 3,8에서 세포의 자기 갱신 상태를 모니터링하는 데 사용할 수 있습니다. RNAi와 결합, Oct4GiP 기자 분석은 신속 ES 세포 유지 및 자기 갱신 3,8의 규제를 파악하고 공부하는 데 사용할 수 있습니다. 자기 갱신을 시금위한 다른 방법과 비교했을 때, 그것은 더 편리하고 민감한, 양적, 그리고 낮은 비용입니다. 이러한 높은 처리량 화면 또는 유전자 epistasis 분석과 같은 대규모 연구 또는 접시 384 - 잘 - 그것은 96에 실시 할 수 있습니다. 마지막으로, 다른 혈통 특정 기자의 ES 세포 라인을 사용하여, 우리가 여기서 설명하는 분석도 ES 세포 분화 동안 운명 사양을 공부하고 수정할 수 있습니다.

Watch this Scientific Journal Video about Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal at JoVE.com

Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal

우리는 신속하게 식별하고 마우스 배아 줄기 세포 유지 및 자기 갱신을 조절 유전자를 특성화하기 위해 형광 기자 분석을 설명합니다.

마우스 배아 줄기 세포 유지 보수 및 자체 갱신을 조절 유전자를 공부하는 Oct4GiP 리포터 분석

Pluripotency and self-renewal are two defining characteristics of embryonic stem cells (ES cells). Understanding the underlying molecular mechanism will ...

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Research

JoVE Journal

Biology

10.4K 조회수.  National Institute of Environmental Health Sciences. 우리는 신속하게 식별하고 마우스 배아 줄기 세포 유지 및 자기 갱신을 조절 유전자를 특성화하기 위해 형광 기자 분석을 설명합니다.

비디오: Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

Murine 배아 줄기 세포로부터 조혈 줄기 세포의 유도

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

연구

생물학

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

Immunosurgery에 의해 인간 배아 줄기 세포의 유도

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative medicine. However, direct injection of hESCs, and cells differentiated from hESCs, into living organisms has thus far been hampered by significant cell death, teratoma formation, and host immune rejection. Understanding the in vivo hESC behavior after transplantation requires novel imaging techniques to longitudinally monitor hESC localization, proliferation, and viability. Molecular imaging has given investigators a high-throughput, inexpensive, and sensitive means for tracking in vivo cell proliferation over days, weeks, and even months. This advancement has significantly increased the understanding of the spatio-temporal kinetics of hESC engraftment, proliferation, and teratoma-formation in living subjects.

A major advance in molecular imaging has been the extension of noninvasive reporter gene assays from molecular and cellular biology into in vivo multi-modality imaging platforms. These reporter genes, under control of engineered promoters and enhancers that take advantage of the host cell s transcriptional machinery, are introduced into cells using a variety of vector and non-vector methods. Once in the cell, reporter genes can be transcribed either constitutively or only under specific biological or cellular conditions, depending on the type of promoter used. Transcription and translation of reporter genes into bioactive proteins is then detected with sensitive, noninvasive instrumentation (e.g., CCD cameras) using signal-generating probes such as D-luciferin.

To avoid the need for excitatory light to track stem cells in vivo as is required for fluorescence imaging, bioluminescence reporter gene imaging systems require only an exogenously administered probe to induce light emission. Firefly luciferase, derived from the firefly Photinus pyralis, encodes an enzyme that catalyzes D-luciferin to the optically active metabolite, oxyluciferin. Optical activity can then be monitored with an external CCD camera. Stably transduced cells that carry the reporter construct within their chromosomal DNA will pass the reporter construct DNA to daughter cells, allowing for longitudinal monitoring of hESC survival and proliferation in vivo. Furthermore, because expression of the reporter gene product is required for signal generation, only viable parent and daughter cells will create bioluminescence signal; apoptotic or dead cells will not. 

In this video, the specific materials and methods needed for tracking stem cell proliferation and teratoma formation with bioluminescence imaging will be described.

With the growing interest in stem cell therapies, molecular imaging techniques are ideal for monitoring stem cell behavior after transplantation. Luciferase reporter genes have enabled non-invasive, repetitive assessment of cell survival, location, and proliferation in vivo. This video will demonstrate how to track hESC proliferation in a living mouse.

체외에서 인간 배아 줄기 세포의 생체내의 Bioluminescence 리포터 진 영상에

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative ...

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

Culture and Maintenance of Human Embryonic Stem Cells

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

인간 배아 줄기 세포의 문화 및 유지 관리

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

The use of SC1 (Pluripotin) to Support mESC Self-renewal in the Absence of LIF

Mouse embryonic stem (ES) cells are conventionally cultured with Leukemia Inhibitory Factor (LIF) to maintain self-renewal.1 However, LIF is expensive and activation of the LIF/JAK/STAT3 pathway is not absolutely required to maintain the self-renewal state.2 The SC1 small molecule may be an economical alternative to LIF. SC1 functions through dual inhibition of Ras-GAP and ERK1.3 Illustration of its mechanism of action makes it a useful tool to study the fundamental molecular mechanism of self-renewal. Here we demonstrate the procedure for culturing mouse ES cells in the presence of SC1 and show that they are able to maintain self-renewal in the absence of LIF. Cells cultured with SC1 showed similar morphology compared to cells maintained with LIF. Both exhibited typical mouse ES morphology after five passages. Expression of typical pluripotency markers (Oct4, Sox2, Nanog, and SSEA1) was observed after five passages in the presence of SC1. Furthermore, SC1 caused no overt toxicity on mouse ES cells.

The use of SC1 Pluripotin to Support mESC Self-renewal in the Absence of LIF

SC1 functions through dual inhibition of Ras- GAP and ERK1. We tested the function of SC1 in supporting mouse ES cell self-renewal in the absence of LIF and showed that SC1 is able to maintain self-renewal of mouse ES cell cultures.

난생의 부재에서 자기 갱신 mESC를 지원하기 위해 SC1 (Pluripotin)의 사용

Mouse embryonic stem (ES) cells are conventionally cultured with Leukemia Inhibitory Factor (LIF) to maintain self-renewal.1 However, LIF is expensive and ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

절연 및 마우스 배아 새싹 세포의 유도

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching morphogenesis. We have developed the limb skin vasculature model to study vascular development in which a pre-existing primitive capillary plexus is reorganized into a hierarchically branched vascular network. Whole-mount confocal microscopy with multiple labelling allows for robust imaging of intact blood vessels as well as their cellular components including endothelial cells, pericytes and smooth muscle cells, using specific fluorescent markers. Advances in this limb skin vasculature model with genetic studies have improved understanding molecular mechanisms of vascular development and patterning. The limb skin vasculature model has been used to study how peripheral nerves provide a spatial template for the differentiation and patterning of arteries. This video article describes a simple and robust protocol to stain intact blood vessels with vascular specific antibodies and fluorescent secondary antibodies, which is applicable for vascularized embryonic organs where we are able to follow the process of vascular development.

We introduce a whole-mount immunohistochemistry and laser scanning confocal microscopy with multiple labelling for analyzing intricate vascular network formation in mouse embryonic limb skin.

배아 림브 스킨 Vasculature에 대한 전체 마운트 Immunohistochemical 분석 : 엠브료에 혈관 분기 Morphogenesis를 공부하는 모델 시스템

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching ...

Modified Mouse Embryonic Stem Cell based Assay for Quantifying  Cardiogenic Induction Efficiency

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways. One of the hurdles to its understanding has been the varied methods in correlating changes of key signaling events to differentiation efficiency. We describe here the use of a mouse embryonic stem (ES) cell based assay to identify critical time windows for Wnt/&beta;-catenin and BMP signal activation during cardiogenic induction. By scoring for contracting embryonic bodies (EBs) in a 96-well plate format, we can quickly quantify cardiogenic efficiency and identify crucial time windows for Wnt/&beta;-catenin and BMP signal activation in a time course following specific modulator treatments. The principal outlined here is not limited to cardiac induction alone, and can be applied towards the study of many other cell lineages. In addition, the 96-well format has the potential to be further developed as a high throughput, automated assay to allow for the testing of more sophisticated experimental hypotheses.

We describe the use of a mouse ES cell based assay to identify critical time windows for Wnt/&#946;-catenin and BMP signal activation during cardiogenic induction. The method provides a standardized platform that reliably quantifies cardiogenic efficiency, and it is applicable to the study of other cell lineages.

Cardiogenic 유도 효율을 Quantifying에 대한 수정 마우스 배아의 줄기 세포 기반 어세이

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways.  One of the hurdles ...

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo. Here we present a method to perform ChIP using a dissociated embryonic day 8.5 (E8.5) embryo. Sheared chromatin from a single E8.5 embryo can be divided into up to five aliquots, which allows the investigator sufficient material for controls and for investigation of specific protein:chromatin interactions.
We have utilized this technique to begin to document protein:chromatin interactions during the specification of tissue-specific gene expression programs. The heterogeneity of cell types in an embryo necessarily restricts the application of this technique because the result is the detection of protein:chromatin interactions without distinguishing whether the interactions occur in all, a subset of, or a single cell type(s). However, examination of tissue-specific genes during or following the onset of tissue-specific gene expression is feasible for two reasons. First, immunoprecipitation of tissue specific factors necessarily isolates chromatin from the cell type where the factor is expressed. Second, immunoprecipitation of coactivators and histones containing post-translational modifications that are associated with gene activation should only be found at genes and gene regulatory sequences in the cell type where the gene is being or has been activated. The technique should be applicable to the study of most tissue-specific gene activation events.
In the example described below, we utilized E8.5 and E9.5 mouse embryos to examine factor binding at a skeletal muscle specific gene promoter. Somites, which are the precursor tissues from which the skeletal muscles of the trunk and limbs will form, are present at E8.5-9.54,5. Myogenin is a regulatory factor required for skeletal muscle differentiation 6-9. The data demonstrate that myogenin is associated with its own promoter in E8.5 and E9.5 embryos. Because myogenin is only expressed in somites at this stage of development 6,10, the data indicate that myogenin interactions with its own promoter have already occurred in skeletal muscle precursor cells in E8.5 embryos.

We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development.

초기 마우스 배아를 사용하는 조직 특정 유전자에 대한 염색질의 Immunoprecipitation 분석

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

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

유도 만능의 줄기 세포에서 파생 마우스의 생성

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...