我们感谢阅读和编辑稿件:布拉德Lackford。这项研究是由美国国家环境健康科学，健康院内研究计划Z01ES102745国家研究院​​（生长激素）研究所的支持。

1。 Oct4GiP小鼠胚胎干细胞维持请提供由奥斯汀·史密斯博士Oct4GiP细胞。他们来自129/Ola携带Oct4的4,5转基因GFPiresPac小鼠。他们均保持在明胶涂层的组织培养皿中的ESGRO完整加克隆的高档中型（Millipore公司），或在的M15培养基:DMEM培养基（Invitrogen公司）15％胎牛血清，1000 U / ml的ESGRO（Millipore公司），1个非补充人体必需的氨基酸（Invitrogen公司），1X EmbryoMax Nucleasides（Millipore公司），β-巯基乙醇和10μm。 <ol><li>用0.1％明胶（Sigma公司）（明胶溶液0.1毫升/厘米2）在室温下30分钟的外套板。 </li><li>明胶涂层钢板的板Oct4GiP细胞〜2×10 4 /厘米2。 </li><li>分裂的细胞，每两天0.05％的胰蛋白酶。 </li></ol> 2。 Oct4GiP细胞在siRNA转染 Oct4GiP记者分析是最convenien的TLY进行了明胶涂层平底的M15培养基在96 - 或384孔板（Corning或BD）。整个过程的概述图1。 <ol><li> siRNA的脂质复合物的组装: <ol><li>在96孔板转染，组装在U型底部的siRNA脂质复合物的96孔板。 </li><li>在每口井，混合10，微升OptiMEM（Invitrogen公司）和0.3μL脂质体2000（Invitrogen公司），并在室温下孵育5分钟。 </li><li>加入5 pmol的siRNA的的脂质OptiMEM混合物孵育15分钟。 siRNA的脂质比例是100 pmol:6ul的。 </li><li>明胶涂层平底板与多通道移液器在OptiMEM从U型底板的siRNA脂质复合物转移。 </li><li>在384孔板转染，准备使用0.1μL脂质体2000年和2 pmol的siRNA 10μLOptiMEM中的siRNA脂质复合物。 </li></ol></li></ol>注:最后的siRNA浓度转染是50纳米。开展3-4生物复制为每个转染。设立在U型底板和分装成平底明胶涂层板的siRNA脂质复合物的主混合物。 <ol start="2"><li> Oct4GiP细胞接种和转染: <ol><li>在96孔板转染，收集Oct4GiP细胞和悬浮在M15的新鲜中等9×10 4细胞/毫升。 </li><li>加入100μL的细胞悬液，以每口井使用多通道移液器。 </li><li>混合，分装前的siRNA脂质复合物在井pipeting和3-5倍的细胞悬液。更改为不同的siRNA转染的技巧。 </li><li>在384孔板，重悬Oct4GiP细胞转染至6×10 4细胞/毫升，分装30μL细胞悬液，以每口井。 </li></ol></li></ol>注:最佳细胞接种密度通常为3×10 5细胞/厘米2，但为mAY需要进一步优化，极大地影响细胞的生长或活力的siRNAs。电镀密度低，会导致在转染细胞存活率不佳。电镀密度高，将导致高的背景下，由于高细胞融合诱导分化。如果必要，测试镀层密度2-4×10 5细胞/平方厘米之间。 <ol start="3"><li>介质的变化: <ol><li>删除旧的介质，采用多渠道吸引器（康宁）或真空棒（VP-科学）。要小心不要划伤板块底部的细胞。 </li><li>喂新鲜的ES细胞培养基，使用多通道移液器每天96孔板和384孔板30μL（100μL）的细胞。 </li></ol></li></ol> 3。流式细胞仪分析<ol><li>细胞分离: <ol><li>染后四天，除去介质，采用多渠道吸引器（康宁）或真空棒（VP-科学）。 </li><li>对于96孔板，一次冲洗细胞与100μL PBS。 </li><li>加入25微升0.25％胰蛋白酶，每口井使用多通道移液器。 </li><li>为偶尔搅拌5分钟，室温孵育和目视检查，以确保细胞完全脱离。 </li><li> 90 PBS液加入含10％胎牛血清灭活胰蛋白酶，分解成单个细胞悬液，重复移液的细胞。 </li><li> 384孔板，撇清与10μL胰蛋白酶和淬火与30μl的PBS含10％胎牛血清的细胞。 </li></ol></li><li>流式细胞仪分析: <ol><li>配备的超导装置或其他类似配备流式细胞仪（如Accuri或Intellicyt），屋宇署LSRII流式细胞仪分析GFP荧光。 </li><li>使用高温超导高通量的模式，并分析10μL细胞悬液。设置计数门槛1.0×10 4细胞。 </li><li>调整PMT电压和阈值来捕获细胞，在前进与侧散射的情节。闸门在FORW的活细胞人口ARD与侧散点图。 </li><li>创建一个GFP的通道直方图的阴谋。设置在GFP通道的门，使〜10％的细胞似乎是在模拟或控制的siRNA转染细胞GFP阴性。 </li><li>确定从每个治疗％GFP阴性细胞:％=％的GFP阴性细胞分化的细胞。比较％之间的siRNA实验和控制使用双尾t检验转染分化的细胞，并取得积极的命中p值&lt;0.01。 </li></ol></li></ol> 4。代表结果 OCT4，NANOG和SOX2基因维持ES细胞的自我更新6,7,9-11中发挥关键作用。 图2显示，Oct4GiP记者分析可以很容易地检测沉默在这些因素造成的分化Oct4GiP胚胎干细胞。 图2A显示Oct4GiP ES细胞保持胚胎时，GFP阳性<html> 图2B细胞显示正向与侧面的散点图和GFP的控制或Oct4的-siRNA的转染细胞Oct4GiP通道的直方图。这是必要的门在前进 - 侧散点图活细胞，死细胞和碎片GFP阴性，并会增加背景。另一方面，偶极歧视，以排除可能的细胞团块并不总是需要。在控制siRNA转染细胞，细胞的绝大多数应该是GFP阳性。如果明显GFP负人口目前在控制siRNA转染井，开始Oct4GiP细胞或转染过程可能已被破坏的结果可能是无法解释的。 图2C显示条形图％分化细胞（％=％GFP阴性细胞分化的细胞）控制，OCT4，Nanog的，和Sox2 siRNA转染细胞。 <html> d/3987/3987fig1.jpg"/&gt; 图1。纲要的Oct4GiP记者分析 。 <img alt="图2" src="/files/ftp_upload/3987/3987fig2.jpg" /> 图2。 Oct4GiP记者分析可以检测胚胎干细胞分化引起Nanog的，OCT4，SOX2沉默。一）E14Tg2a（野生型，黑线）和Oct4GiP细胞（绿线），流式细胞仪分析。 GFP的通道直方图显示，Oct4GiP细胞GFP阳性。）Oct4GiP细胞转染控制或Oct4的-siRNA的96孔板和流式细胞仪分析转染后4天。提出与绿色荧光蛋白转染细胞通道一侧的散点图和柱状图显示C）Oct4GiP细胞转染控制，NANOG，Oct4的，或SOX2的siRNA。 ％分化的细胞被确定从％的GFP阴性细胞转染后4天，并绘制了作为意味着+ / - 平均值的标准误差（N = 4）。F ="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/3987/3987fig2large.jpg"目标="_blank"&gt;点击这里查看大图。

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没有利益冲突的声明。

我们上面描述的Oct4GiP记者分析，可以定量衡量的自我更新与分化程度。其他可用的方法，如12基于形态和增殖/活力检测，比较，它提供了更高的灵敏度和吞吐量，以及胚胎干细胞状态的一个更直接的测量。因此，非常适合大型屏幕和遗传上位性分析。事实上，我们和其他人已成功地用于全基因组RNAi屏幕3,8 Oct4GiP记者分析。然而，所有实验一样，它也有其局限性。它只能被用来研究的基因，直接或间接的调控Oct4的启动子活性，它可能会错误地识别影响GFP的表达，稳定，功能转录后的基因。因此，额外的检测或辅助屏幕，如碱性磷酸酶染色13（Millipore公司，SCR004）和免疫荧光染色或定量RT多能性标志物-聚合酶链反应3,8,12,14需要确认这种方法得到的结果。 Oct4GiP记者分析，依靠高效的基因沉默。有效的siRNA和siRNA的转染效率的检测成功的关键。虽然我们只描述了记者与siRNA转染法的使用，也可以进行检测的DNA转染沉默或基因过度表达的兴趣。 DNA转染效率通常是较低的siRNA，并可能降低强度的表型。 除了在本协议中所述，Oct4GiP记者分析，可以识别和表征基因负调节自我更新以及修改。在这种情况下，可以转染细胞的siRNA，并在分化条件下培养。沉默的自我更新的负调控将加强自我更新和维持GFP的表达，可以是detected同样在当前协议的流式细胞仪。 除了​​Oct4GiP记者的细胞，其他记者使用胚胎干细胞特异性启动子，如在Nanog的绿色荧光蛋白15-18（Millipore公司，SCR089）和Rex1-GFP 19细胞的细胞株，也已产生。它们也可以用来研究胚胎干细胞自我更新和维护，使用同样的策略。然而，这些胚胎干细胞标记基因启动子的活性和特异性，因为有明显的区别，这些记者的细胞株有不同的行为在背景水平和灵敏度方面，从而将相得益彰。最后，通过使用其他系特定记者ES细胞系，我们的战略可以修改研究的胚胎干细胞的命运，规范和促进ES细胞在体外哺乳动物早期发育模型的使用。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> ESGRO完整加克隆的高档中型</td><td> Millipore公司</td><td> SF001-500P </td></tr><tr><td> DMEM培养液（高糖1X） </td><td> Invitrogen公司</td><td> 11965 </td></tr><tr><td> 0.25％胰蛋白酶EDTA </td><td> Invitrogen公司</td><td> 25200 </td></tr><tr><td>脂质体2000 </td><td> Invitrogen公司</td><td> 1001817 </td></tr><tr><td> OPTI-MEM中（降低血清培养基） </td><td> Invitrogen公司</td><td> 31985 </td></tr><tr><td> ESGRO mLIF（7 units/1ml） </td><td> Millipore公司</td><td> DAM1776540 </td></tr><tr><td> NEAA的MEM（非必需氨基酸） </td><td> Invitrogen公司</td><td> 11140 </td></tr><tr><td>胚胎干细胞的100倍核苷</td><td> Millipore公司</td><td> 10620-1 </td&gt; </tr><tr><td> 2 - 巯基乙醇</td><td>西格玛</td><td> M7522-100ML </td></tr><tr><td> ES合格的胎牛血清</td><td> Invitrogen公司</td><td> 10437 </td></tr><tr><td> Nanog基因的siRNA </td><td> Invitrogen公司</td><td> MSS231181 </td></tr><tr><td> Oct4的RNA干扰</td><td> Dharmacon的</td><td>的D-046256-02 </td></tr><tr><td> SOX2的siRNA </td><td> Dharmacon的</td><td>的M-058489-01 </td></tr></tbody></table>对照siRNA:siRNA双链针对萤火虫荧光素酶（5&#39;-CGTACGCGGAATACTTCGA）由Dharamcon的合成。

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

两个定义特征的胚胎干细胞（ES细胞）的全能性和自我更新。了解潜在的分子机制，将大大方便使用胚胎干细胞的发育生物学研究，疾病模型，药物发现和再生医学（1,2审查）。 为了加快胚胎干细胞的维持和自我更新的小说监管的识别和表征，我们开发了一种基于荧光记者分析定量测量使用Oct4GiP细胞3在小鼠胚胎干细胞的自我更新的状态。 Oct4基因启动子区域4,5的控制下，Oct4GiP细胞表达绿色荧光蛋白（GFP）。 Oct4的需要为ES细胞的自我更新，高度在ES细胞中表达，并迅速下调在分化6,7。因此，GFP的表达和记者的细胞荧光相关忠实与胚胎干细胞的身份5，荧光激活细胞分选仪（FACS）分析可以用来密切注视3,8在单细胞水平细胞的自我更新的状态。 用RNAi耦合，Oct4GiP的记者法可用于快速识别和研究的胚胎干细胞维持和自我更新3,8监管。化验自我更新的其他方法相比，它更方便，灵敏，定量，成本更低。它可进行96 - 或384孔板为大规模的研究，如高通量筛选或遗传上位性分析。最后，通过使用其他血统的特定记者ES细胞系，我们在这里描述的检测，还可以进行修改，研究胚胎干细胞分化过程中的命运规范。

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

我们描述记者荧光法快速识别和表征基因调控小鼠胚胎干细胞维持和自我更新。

Oct4GiP记者试验研究小鼠胚胎干细胞维持和自我更新的基因调节

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

oct4gip-reporter-assay-to-study-genes-that-regulate-mouse-embryonic

Nanyang Technological University

Research

JoVE Journal

Biology

10.4K 次观看.  National Institute of Environmental Health Sciences. 我们描述记者荧光法快速识别和表征基因调控小鼠胚胎干细胞维持和自我更新。

视频: 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.

从小鼠胚胎干细胞的造血干细胞的推导

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 ...

科研

生物学

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.

人类胚胎干细胞的推导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 ...

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.

文化和维护人类胚胎干细胞

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.

SC1（Pluripotin）使用，以支持在没有LIF的自我更新MESC

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.

小鼠胚胎生殖细胞的分离和推导

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

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation 

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three embryonic germ layers (1). Directed differentiation of hESCs into specific cell types has generated much interest in the field of regenerative medicine (e.g., (2-5)), and methods for determining the in vivo fate of selected or manipulated hESCs are essential to this endeavor. We have adapted a highly efficient teratoma formation assay for this purpose. A small number of specifically selected hESCs is mixed with undifferentiated wild type hESCs and Phaseolus vulgaris lectin to form a cell pellet. This is grafted beneath the kidney capsule in an immunodeficient mouse. As few as 2.5 x 105 hESCs are needed to form a 16 cm3 teratoma within 8-12 weeks. The fate of the originally selected hESCs can then be determined by immunohistochemistry. This method provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

Directed differentiation of hESCs into specific cells has generated much interest in regenerative medicine. We provide a concise, step-by-step protocol for determining the in vivo fate of selected hESCs that provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

人类胚胎干细胞畸胎瘤形成命运的映射

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three ...

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.

全安装胚胎肢体皮肤血管的免疫组织化学分析:一个模型系统，研究在胚胎血管分支形态发生

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.

基因小鼠胚胎干细胞的基础含量为心源性诱导效率量化

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.

使用早期的小鼠胚胎组织特异性基因的染色质免疫沉淀检测

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.

诱导多能干细胞来源于小鼠的生成

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