CACW得到医学研究理事会博士生的支持。 GMF部分由医学研究委员会新研究者奖（MR / N000609 / 1）和Tenovus苏格兰研究资助部分支持。

小分子激酶抑制剂库的整理<ol><li>来自可公开获得的激酶抑制剂收集物的分页抑制剂文库， 例如来自哈佛医学院的基于综合网络的细胞标记库（LINCS），228种有效和选择性激酶抑制剂的靶向收集物（http://lincs.hms.harvard.edu） ，和蛋白激酶抑制剂组（PKIS），由工业研究人员（https://www.ebi.ac.uk/chembldb/extra/PKIS）组装的376化合物收集品13 。或者，从市售化合物组装定制的激酶抑制剂收集物。 注意:在我们的实验室，我们汇集了一系列72种有效和选择性的工具激酶抑制剂，涵盖51种主要靶向激酶，所有这些都可从商业来源获得。 </li><li>将激酶抑制剂装配到96孔板中，以实现多种浓度的最佳高通量处理（使用1，0.1 and 0.01mM）。包括在含有DMSO的每个平板对照孔和参考对照抑制剂PD173074（FGFRi）和Ruxolitinib（JAKi）中，其分别促进幼稚和引发的多能态。在电子表格或类似软件中注释。 </li></ol> 2. mESC培养条件和筛选程序<ol><li>通过向10厘米平板中加入5毫升0.1％（w / v）明胶制备0.1％明胶包被的10厘米培养皿，并在室温下孵育5分钟。吸出明胶，让板干燥2分钟。 </li><li>在含有100ng / mL GST-LIF，10％胎牛血清和5％敲除血清的标准mESC培养基（参见材料表 ）中，在37℃5％CO 2的0.1％明胶包被的10cm培养皿上培养任何标准mESC系替代。每天更换培养基，通过mESCs，每隔约80％汇合，稀释1:5-1:10。 </li><li>通过mESCs，吸出培养基并用5 mL o洗涤f磷酸盐缓冲盐水（PBS）。 </li><li>加入每平板mESCs 1 mL胰蛋白酶EDTA（0.05％胰蛋白酶，0.02％EDTA），37℃孵育10 min。 </li><li>将胰蛋白酶处理的细胞重悬于4mL的mESC培养基中，并以1,200rpm离心5分钟。 </li><li>在5mL的mESC培养基中彻底重悬细胞沉淀，上下移液以产生单细胞悬液。 </li><li>通过将10μL细胞悬浮液和10μL台盼蓝（0.4％）组合计数细胞。放入细胞计数室或使用血细胞计数器和光学显微镜。 </li><li>将3×10 3个 mESC种子加入到0.1％明胶包被的96孔板中，最终体积为100μL的培养基，使用多通道移液管。 </li><li>使用多通道移液器，以1:100稀释（1μL抑制剂加入100μL培养基）中应用激酶抑制剂。轻轻移液培养基以混合抑制剂和细胞悬浮液，然后使细胞在组织培养罩中沉降1小时以确保相等分布acro电镀表面。培养细胞48h，不改变培养基。 注意:1 / 0.1 / 0.01mM的储备板将分别给出10/1 /0.1μM的最终抑制剂浓度。我们建议以1μM的最终浓度启动荧光屏，以确保有效抑制主要靶激酶，同时最大限度地减少目标效应。 </li></ol>激酶抑制剂筛选分析<ol><li>使用多通道吸气器和移液管在200μLPBS中洗涤96孔mESC平板。 </li><li>在150μL裂解缓冲液（20mM Tris pH7.4,150mM NaCl，1mM EDTA，1％NP-40（v / v），0.5％脱氧胆酸钠（w / v），10mMβ-甘油磷酸钠，10mM焦磷酸钠，1mM NaF，2mM Na 3 VO 4 ，完全蛋白酶抑制剂鸡尾酒片）。 </li><li>通过在V底96孔板中以1,500×g离心30分钟澄清提取物。 </li><li>将100μL上清液固定在硝基上lulose膜使用96孔真空斑点印迹歧管。 </li><li>干燥膜，并用40毫升Ponceau S染色，以确保一致的转印。 </li><li>用TBST洗涤膜，并在TBST / 3％（w / v）牛奶中封闭。 </li><li>以1:1,000（v / v）稀释于TBST / 3％（w / v）奶粉中的Nanog和Dnmt3b抗体孵育过夜。 </li><li>在TBST中以3×10分钟洗涤膜，并在TBST / 3％（w / v）牛奶中稀释1:10（v / v）的30mL二级抗体中孵育。 </li><li>开发使用数字免疫印迹成像系统。 注意:如果可能，使用定量荧光免疫印迹检测Nanog和Dnmt3b。然而，通过荧光免疫印迹对Dnmt3b的背景信号太高。因此，使用800nm荧光抗兔（Nanog）和抗小鼠HRP（Dnmt3b）二抗。 </li></ol> 4.筛选数据分析和抑制剂作为真菌多能调节剂的验证<ol><li>使用免疫印迹分析软件量化Nanog和Dnmt3b信号，并将值导入电子表格或类似软件。与DMSO对照相比，计算所有样品的Nanog:Dnmt3b比例。此外，确定总Nanog和Dnmt3b信号，以确保激酶抑制剂通过改变mESC活力不影响Nanog:Dnmt3b比例。 </li><li>使用排列的Nanog:Dnmt3b比率来鉴别促进幼稚多能性（Nanog:Dnmt3b比高于对照）和引发多能性（Nanog:Dnmt3b比低于对照）的抑制剂。将截止值设置为与对照值的2倍偏差，并过滤掉产生总Nanog + Dnmt3b信号的激酶抑制剂50％的DMSO对照值，以产生稳定初始（高Nanog:Dnmt3b比）和引发（低Nanog:Dnmt3b比）多能态的激酶抑制剂的高置信列表。 </li><li>通过在2mL培养基中每6孔以2×10 5个细胞接种mESC来验证激酶抑制剂，并在1μM下加入激酶抑制剂48小时，而不改变培养基。 </li><li> Lyse mESCs在裂解缓冲液中，通过常规SDS-PAGE免疫印迹分析Nanog和Dnmt3b表达。 </li><li>通过SDS-PAGE免疫印迹进一步验证初始多能性标记Klf2和Klf4以及Oct4，以确保48小时抑制剂治疗后保持多能性。 </li></ol>

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作者声明本研究没有产生利益冲突。

在这里，我们展示了一种广泛可用的方法来探索激酶信号通路在调节初始化多能转换中的作用。这解决了ESC字段中的一个关键问题。虽然高通量基因组学和转录组学方法常规用于鉴定初始和引发多能状态的关键转录调控因子，但是阐明多能性信号传导网络已被证明是具有挑战性的。我们现在提供一种灵活的策略，采用化学抑制剂来鉴定在mESCs中控制初始化多能转化的激酶。这取决于大多数研究细胞信号传导和ESC生物学实验室的简单设备，试剂和材料。对于这个平台的成功至关重要的是我们开发了一个强大的测定来量化初始和引发多能态之间的转变。建立该测定需要对细胞平台进行重大的迭代修饰抑制剂孵育时间和免疫印迹条件。 小分子方法在治疗性ESC应用15,16中得到合理使用。小分子筛选的主要优点是设计和/或修饰工具化合物以有效的细胞摄取。转染效率并不局限于此，不需要使用慢病毒等危险递送系统。此外，抑制剂经常抑制激酶家族内的多种同种型（ 例如 Fgfr1-4，Jak1-3），其克服阻碍基因/基因组干扰技术如RNAi和CRISPR / Cas9的功能冗余。由于学术和药物药物发现计划中提供了更有效和更有选择性的工具化合物，因此研究较少且知之甚少的激酶将被推向ESC研究的前沿。因此，我们建议这样高的粗略筛选方法将为核心ESC监管网络开辟新的研究途径。 该技术的一个小小的限制是即使是最有效和选择性的小分子抑制剂也可以在体内接触和抑制多种不相关的激酶。然而，日益全面的激酶抑制剂分析数据可以容易地鉴定激酶抑制剂的功能靶点（http://lincs.hms.harvard.edu/kinomescan; http://www.kinase-screen.mrc.ac.uk/kinase -inhibitors）。最后，原则上我们的平台可以应用于询问可获得高质量免疫印迹抗体的任何小分子收集和/或细胞测定。这将允许研究多能调节系统的多能调节和促进鉴定改变不同细胞过程的小分子抑制剂。具体来说，我们设想应用新兴的小分子收集物，如epigene由结构基因组学联盟（http://www.thesgc.org/chemical-probes/epigenetics）开发的tic探针有潜力揭示更多新型多能转录调控因子。

胚胎干细胞（ESCs）具有自我更新或分化成成体内任何细胞类型的能力，这种现象称为多能性1 。最近的证据表明，存在发育不同的多能性状态，称为"初始"和"初始化"多能性2 。初始胚胎干细胞代表与植入前胚胎相似的发育状态3 。相比之下，引发多能的胚胎干细胞准备退出多能性并分化成专门的胚胎谱系4,5 。 原始的多能性状态以不同的基因调控网络为特征。最初的多能性的特征在于表达关键的多能转录因子，如Nanog，Krueppel样转录因子（Klfs），Rex2和Esrrb <supclass ="xref"&gt; 6。在小鼠ESCs（mESCs）中，引发多能性的特征在于初始标记物的表达减少，特异性基因表达特征包括从头DNA甲基转移酶Dnmt3b 7 。 在体内 ，引发多能的植入后表皮细胞干细胞（EpiSCs） 4,5还附加表达Epiblast标记Fgf5和谱系引发标志物如Brachyury 8 。 mESCs提供了一种易于理解的模型，用于探索在体外控制初始化多能转录的机制 。当在白血病抑制因子（LIF）和胎牛血清（FBS）中培养时，mESCs在初始和引发的多能态之间经历动态过渡9,10 。 LIF-Jak-Stat3信号传导功能，促进幼稚基因调控网络11 </而通过成纤维细胞生长因子4（Fgf4）Erk1 / 2通路的自分泌信号驱动转变到引发状态12 。然而，系统地评估蛋白激酶在指定明显多能状态中的作用仍然是一个挑战。 在这里，我们描述了一个定量和可扩展的平台，通过该平台为初始化多能转换的激酶调节剂进行目标小分子筛选。我们使用简单的mESC培养条件和标准试剂，材料和设备来发现和验证激酶抑制剂，迄今为止尚未得到稳定的初始多能性的能力。此外，我们还讨论了该技术的潜在扩展应用，包括筛选其他小分子系列，例如靶向表观遗传调节因子的新兴抑制剂。

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">mESC media</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CCE mESCs</td>
			<td style="width:71px;">ATCC</td>
			<td style="width:119px;">ATCC&#160;SCRC-1023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM Media</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">11965092</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">L-Glutamine</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">25030081</td>
			<td>Final: 2mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Penicillin/Streptomycin</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">15140122</td>
			<td>Final: 50U/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2-mercaptoethanol</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">M6250</td>
			<td>Final: 0.1mM</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Leukemia Inhibitory Factor (GST fusion)</td>
			<td style="width:71px;">MRC-PPU reagents and services</td>
			<td style="width:119px;"></td>
			<td>Final: 100ng/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Leukemia Inhibitory Factor</td>
			<td style="width:71px;">Thermo</td>
			<td style="width:119px;">PMC9484</td>
			<td>Final: 100ng/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Non-Essential Amino Acids</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">11140050</td>
			<td>Final: 0.1mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium Pyruvate</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">11360070</td>
			<td>Final: 1mM</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">KnockOut Serum Replacement</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">10828-028</td>
			<td>Final: 5%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Defined Fetal Bovine Serum</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:119px;">10703464</td>
			<td>Final: 10%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
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			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gelatin from porcine skin</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Nunc MicroWell 96-Well Microplates</td>
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			<td height="42" style="height:42px;width:216px;">DPBS, no calcium, no magnesium</td>
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			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">V-bottom 96 well plates</td>
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			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1% NP-40</td>
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			<td height="21" style="height:21px;width:216px;">Sodium Orthovanadate</td>
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			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Roche Complete Protease Inhibitor Cocktail Tablets</td>
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		</tr>
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			<td height="21" style="height:21px;width:216px;">Name</td>
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			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">P1379-1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Milk Powder</td>
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			<td height="21" style="height:21px;width:216px;">Western Blotting</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
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		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Protran BA 85 Nitrocellulose 0.45uM&#160; Pore size (20 X 20cm Sheets) (25 Sheet)</td>
			<td style="width:71px;">GE</td>
			<td style="width:119px;">10401191</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ponceau S</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">P7170-1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name</td>
			<td style="width:71px;">Company</td>
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			<td height="21" style="height:21px;width:216px;">Laboratory Equipment</td>
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			<td style="width:119px;"></td>
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			<td height="21" style="height:21px;width:216px;">Minifold I System</td>
			<td style="width:71px;">GE</td>
			<td style="width:119px;">10447850</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ChemiDoc imager</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LiCor Odyssey Imager</td>
			<td style="width:71px;">LiCor</td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name</td>
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			<td height="21" style="height:21px;width:216px;">Antibodies</td>
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			<td height="21" style="height:21px;width:216px;">Anti-mouse Nanog</td>
			<td style="width:71px;">Reprocell</td>
			<td style="width:119px;">RCAB001P</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-Dnmt3b</td>
			<td>Imgenex</td>
			<td>IMG-184A</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">IRDye 800CW Donkey anti-Rabbit</td>
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			<td style="width:119px;">926-32213</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Anti-mouse-HRP</td>
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			<td>7076S</td>
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			<td height="21" style="height:21px;width:216px;">Anti-Klf2</td>
			<td style="width:71px;">Millipore</td>
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			<td height="42" style="height:42px;width:216px;">Anti-Oct4</td>
			<td style="width:71px;">Santa Cruz</td>
			<td style="width:119px;">sc-5279</td>
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	</tbody>
</table>

a simple method to identify kinases that regulate embryonic stem cell pluripotency by high-throughput inhibitor screening

胚胎干细胞（ESC）可以自我更新或分化成所有的细胞类型，这种现象称为多能性。已经描述了不同的多能性状态，称为"天真"和"初始化"多能性。控制初始化过渡的机制了解甚少。特别是，尽管提供了高质量的工具化合物探索激酶功能的可用性越来越多，但我们对于指定初始和初始多能性状态的蛋白激酶仍然知之甚少。在这里，我们描述了一个可扩展的平台，用于对小鼠胚胎干细胞幼虫初始化多能转化激酶调节剂进行靶向小分子筛选。这种方法利用简单的细胞培养条件和标准试剂，材料和设备来发现和验证激酶抑制剂，迄今为止对多能性的不受欢迎的影响。我们讨论该技术的潜在应用，包括筛选其他小分子诸如越来越复杂的激酶抑制剂和表观遗传工具化合物的新兴文库的集合。

使用本文提供的程序（ 图1 ），我们筛选了228种有效和选择性激酶抑制剂的LINCS文库，以鉴定调节mESC多能性的那些。以1:100稀释浓度0.1mM的浓度制备文库，并在mESCs中最终筛选浓度为1μM。 48小时后，将mESCs溶解，制备提取物用于Nanog和Dnmt3b表达的定量斑点印迹分析的提取物（ 图2 ，上图 ）。为了简洁起见，其他筛选浓度的结果不在此列出。 Nanog:测定每种激酶抑制剂的Dnmt3b比例，以及应用的化合物和2x截止（ 图2 ，底部）。总的Nanog和Dnmt3b相对于对照的信号覆盖在抑制剂排名上并进行0.5x的切断，以允许显示出显着的mESC毒性的抑制剂的分类。选择稳定初始状态的激酶抑制剂te使用常规Nanog / Dnmt3b免疫印迹进行验证（ 图3 ）。这些抑制剂的主要激酶靶标列于表1 。我们还展示了该屏幕的适用性，以确定稳定起始状态的抑制剂14 。 <img alt="图1" src="/files/ftp_upload/55515/55515fig1.jpg" /> 图1:筛选激酶抑制剂的工作流程，调节初始化过渡。 mESC用终浓度为1μM的228复合激酶抑制剂文库处理。制备裂解物并转移到硝酸纤维素膜上用于免疫斑点印迹分析，并测定Nanog和Dnmt3b水平（图中改编自Williams 等人 14 ）。 <a href="http://ecsource.jove.com/files/ftp_upload/55515/55515fig1large.jpg" target="_blank">请点击这里查看al</a><html>这个数字的arger版本。 <img alt="图2" src="/files/ftp_upload/55515/55515fig2.jpg" /> 图2:初始化多能性筛选分析和抑制物鉴定。 （Top）代表性的Nanog和Dnmt3b免疫印迹图像。 （底部）相对于DMSO对照测定每种激酶抑制剂的Nanog和Dnmt3b值，并确定Nanog:Dnmt3b比率和总相对Nanog + Dnmt3b信号，并且抑制剂与DMSO对照相比排名。发现改变Nanog:Dnmt3b比超过或低于DMSO对照的2倍阈值的抑制剂被确定为初始或初始多能性的驱动因素。将低于DMSO对照的阈值设置为损害mESC存活的分类抑制剂。选择进一步验证的抑制剂被突出显示。 （图改编自Williams 等人 14 ）。s / ftp_upload / 55515 / 55515fig2large.jpg"target ="_ blank"&gt;请点击此处查看此图的较大版本。 <img alt="图3" src="/files/ftp_upload/55515/55515fig3.jpg" /> 图3:确认的选定命中化合物的验证。 mESC用1μMAZD4547（选择性FGFR抑制剂）或从图2中筛选鉴定的指示抑制剂处理。通过标准SDS-PAGE和免疫印迹分析测定的Nanog:Dnmt3b水平。相对Nanog:Dnmt3b和Nanog + Dnmt3b的数值如下表所示，稳定初始多能态的抑制剂以绿色突出显示。 <a href="http://ecsource.jove.com/files/ftp_upload/55515/55515fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 复合 </td><td colspan="2"> 主要目标 </td><td> 其他目标 </td></tr><tr><td> AV-951 </td><td colspan="2"> VEGFR </td><td></td></tr><tr><td> HG-6-64-01 </td><td colspan="2"> ABL，BRAF，RET，CSF1R，EGFR，EPHA8，FGFR，FLT3，KIT，LOK，MAP4K1，p38b，MUSK，PDGFR，TAOK3，TNNI3K </td><td></td></tr><tr><td> WH-4-023 </td><td colspan="2"> LCK </td><td> SRC </td></tr><tr><td> R406 </td><td colspan="2"> SYK </td><td></td></tr><tr><td> GW-572016 </td><td colspan="2"> HER2 </td><td> EGFR </td></tr><tr><td> KIN001-244 </td><td colspan="2"> PDK-1 </td><td></td></tr><tr><td> WZ-4-145 </td><td colspan="2"> CSF1R，DDR1，EGFR，TIE1，PDGFR2 </td><td></td></tr><tr><td> WZ-7043 </td><td colspan="2"> CSF1R，DDR1，FGFR和TAO1 </td><td></td></tr><tr><td> GW786034 </td><td colspan="2"> VEGFR1 </td><td> VEGFR2，VEGFR3，＃160; </td></tr><tr><td> AZD-1480 </td><td colspan="2"> JAK2 </td><td></td></tr></tbody></table> 表1:选定的命中化合物及其主要激酶靶标。

Watch this Scientific Journal Video about A Simple Method to Identify Kinases That Regulate Embryonic Stem Cell Pluripotency by High-throughput Inhibitor Screening at JoVE.com

A Simple Method to Identify Kinases That Regulate Embryonic Stem Cell Pluripotency by High-throughput Inhibitor Screening

在这里，我们提出一个定量和可扩展的方案来执行针对性的小分子屏幕，用于初始化多能转换的激酶调节剂。

通过高通量抑制剂筛选鉴定调节胚胎干细胞多能性的激酶的简单方法

Embryonic stem cells (ESCs) can self-renew or differentiate into all cell types, a phenomenon known as pluripotency. Distinct pluripotent states have been ...

a-simple-method-to-identify-kinases-that-regulate-embryonic-stem-cell

Research

JoVE Journal

Developmental Biology

6.4K 次观看.  University of Dundee. 在这里，我们提出一个定量和可扩展的方案来执行针对性的小分子屏幕，用于初始化多能转换的激酶调节剂。 

视频: A Simple Method to Identify Kinases That Regulate Embryonic Stem Cell Pluripotency by High-throughput Inhibitor Screening

Forward Genetic Approach to Uncover Stress Resistance Genes in Mice &#8212; A High-throughput Screen in ES Cells

Phenotype-driven genetic screens in mice is a powerful technique to uncover gene functions, but are often hampered by extremely high costs, which severely limits its potential. We describe here the use of mouse embryonic stem (ES) cells as surrogate cells to screen for a phenotype of interest and subsequently introduce these cells into a host embryo to develop into a living mouse carrying the phenotype. This method provides (1) a cost effective, high-throughput platform for genetic screen in mammalian cells; (2) a rapid way to identify the mutated genes and verify causality; and (3) a short-cut to develop mouse mutants directly from these selected ES cells for whole animal studies. We demonstrated the use of paraquat (PQ) to select resistant mutants and identify mutations that confer oxidative stress resistance. Other stressors or cytotoxic compounds may also be used to screen for resistant mutants to uncover novel genetic determinants of a variety of cellular stress resistance.

Stress resistance is one of the hallmarks for longevity and is known to be genetically governed. Here, we developed an unbiased high-throughput method to screen for mutations that confer stress resistance in ES cells with which to develop mouse models for longevity studies.

正向遗传学的方法来揭开抗逆基因小鼠 - 在ES细胞高通量筛选

Phenotype-driven genetic screens in mice is a powerful technique to uncover gene functions, but are often hampered by extremely high costs, which severely ...

科研

发育生物学

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

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

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

生成CRISPR / Cas9介导的单等位基因缺失来研究小鼠胚胎干细胞功能增强

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

Epigenetic Conversion as a Safe and Simple Method to Obtain Insulin-secreting Cells from Adult Skin Fibroblasts

Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such cells are not readily available, they can be obtained using different approaches that include, among the many, reprogramming and trans-differentiation, with advantages and limitations that are specific of the different techniques. Here a new strategy for the conversion of an adult mature fibroblast into an insulin-secreting cell, arbitrarily designated as epigenetic converted cells (EpiCC), is described. The method has been developed, based on the increasing understanding of the mechanisms controlling epigenetic regulation of cell fate and differentiation. In particular, the first step uses an epigenetic modifier, namely 5-aza-cytidine, to drive adult cells into a "highly permissive" state. It then takes advantage of this brief and reversible window of epigenetic plasticity, to re-address cells toward a different lineage. The approach is designated "epigenetic cell conversion". It is a simple and robust way to obtain an efficient, controlled and stable cellular inter-lineage switch. Since the protocol does not involve the use of any gene transfection, it is free of viral vectors and does not involve a stable pluripotent state, it is highly promising for translational medicine applications.

Here, a new method that allows the conversion of adult skin fibroblasts into insulin-secreting cells is presented. This technique is based on epigenetic conversion, does not involve the use of retroviral vectors nor the acquisition of a stable pluripotent state. It is therefore highly promising for translational medicine applications.

后生转换为安全和简单的方法来获得成人皮肤成纤维细胞胰岛素分泌细胞

Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such ...

Reprogramming Mouse Embryonic Fibroblasts with Transcription Factors to Induce a Hemogenic Program

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We first designed a reporter screen using MEFs from human CD34-tTA/TetO-H2BGFP (34/H2BGFP) double transgenic mice. CD34+ cells from these mice label H2B histones with GFP, and cease labeling upon addition of doxycycline (DOX). MEFS were transduced with candidate TFs and then observed for the emergence of GFP+ cells that would indicate the acquisition of a hematopoietic or endothelial cell fate. Starting with 18 candidate TFs, and through a process of combinatorial elimination, we obtained a minimal set of factors that would induce the highest percentage of GFP+ cells. We found that Gata2, Gfi1b, and cFos were necessary and the addition of Etv6 provided the optimal induction. A series of gene expression analyses done at different time points during the reprogramming process revealed that these cells appeared to go through a precursor cell that underwent an endothelial to hematopoietic transition (EHT). As such, this reprogramming process mimics developmental hematopoiesis "in a dish," allowing study of hematopoiesis in vitro and a platform to identify the mechanisms that underlie this specification. This methodology also provides a framework for translation of this work to the human system in the hopes of generating an alternative source of patient-specific hematopoietic stem cells (HSCs) for a number of applications in the treatment and study of hematologic diseases.

The protocol described here details the induction of a hemogenic program in mouse embryonic fibroblasts via overexpression of a minimal set of transcription factors. This technology may be translated to the human system to provide platforms for future study of hematopoiesis, hematologic disease, and hematopoietic stem cell transplant.

重新编程的小鼠胚胎成纤维细胞转录因子诱导Hemogenic计划

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We ...

A Versatile Mounting Method for Long Term Imaging of Zebrafish Development

Zebrafish embryos offer an ideal experimental system to study complex morphogenetic processes due to their ease of accessibility and optical transparency. In particular, posterior body elongation is an essential process in embryonic development by which multiple tissue deformations act together to direct the formation of a large part of the body axis. In order to observe this process by long-term time-lapse imaging it is necessary to utilize a mounting technique that allows sufficient support to maintain samples in the correct orientation during transfer to the microscope and acquisition. In addition, the mounting must also provide sufficient freedom of movement for the outgrowth of the posterior body region without affecting its normal development. Finally, there must be a certain degree in versatility of the mounting method to allow imaging on diverse imaging set-ups. Here, we present a mounting technique for imaging the development of posterior body elongation in the zebrafish D. rerio. This technique involves mounting embryos such that the head and yolk sac regions are almost entirely included in agarose, while leaving out the posterior body region to elongate and develop normally. We will show how this can be adapted for upright, inverted and vertical light-sheet microscopy set-ups. While this protocol focuses on mounting embryos for imaging for the posterior body, it could easily be adapted for the live imaging of multiple aspects of zebrafish development.

Here, we present a versatile mounting method that allows for the long-term time-lapse imaging of the posterior body development of live zebrafish embryos without perturbing normal development.

斑马鱼发展长期成像的多功能安装方法

Zebrafish embryos offer an ideal experimental system to study complex morphogenetic processes due to their ease of accessibility and optical transparency. ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

人胚胎或诱导多能干细胞对视网膜色素上皮细胞的快速定向分化

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

CARIP-Seq and ChIP-Seq: Methods to Identify Chromatin-Associated RNAs and Protein-DNA Interactions in Embryonic Stem Cells

Embryonic stem (ES) cell self-renewal&#160;and differentiation is governed by extrinsic signals and intrinsic networks of transcription factors, epigenetic regulators, and post-translation modifications of histones that combinatorially influence the gene expression state of nearby genes. RNA has also been shown to interact with various proteins to regulate chromatin dynamics&#160;and gene expression. Chromatin-associated RNA immunoprecipitation (CARIP) followed by next-generation sequencing (CARIP-Seq) is a novel method to survey RNAs associated with chromatin proteins, while chromatin immunoprecipitation followed by next-generation sequencing (ChIP-Seq) is a powerful genomics technique to map the location of post-translational modification of histones, transcription factors, and epigenetic modifiers on a global-scale in ES cells. Here, we describe methods to perform CARIP-Seq and ChIP-Seq, including library construction for next-generation sequencing, to generate global chromatin-associated RNA and epigenomic maps in ES cells.

Here, we describe methods to perform ChIP-Seq and CARIP-Seq, including library preparation for next-generation sequencing, to generate global epigenomic and chromatin-associated RNA maps in ES cells.

CARIP 和芯片序列: 鉴定胚胎干细胞中染色质相关 rna 和蛋白质-DNA 相互作用的方法

Embryonic stem (ES) cell self-renewal&#160;and differentiation is governed by extrinsic signals and intrinsic networks of transcription factors, ...

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

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

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

脊髓培养法研究胚胎鸡组织的发展

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

A Direct and Simple Method to Assess Drosophila melanogaster's Viability from Embryo to Adult

In Drosophila melanogaster, viability assays are used to determine the fitness of certain genetic backgrounds. Allelic variations can result in partial or complete loss of viability at different stages of development. Our lab has developed a method to assess viability in Drosophila from embryo to fully mature adult. The method relies on quantifying the number of progeny present at different stages during development, starting with hatched embryos. After embryos have been quantified, additional stages are counted, including L1/L2, pupae, and mature adults. After all stages have been examined, a statistical analysis such as the chi-square test is used to determine if there is a significant difference between the starting number of progeny (hatched embryos) and later stages culminating in the observed number of adults, thus rejecting or accepting the null hypothesis (that the number of hatched embryos will be equal to the number of larvae, pupae, and adults recorded throughout the stages of development). The primary advantage of this assay is its simplicity and accuracy, as it does not require an embryo rinse to transfer them to the food vial, avoiding losses from technical errors. Although the protocol described here does not directly examine L2/L3 larvae, additional steps can be added to account for these. Comparing the number of hatched embryos, L1, pupae, and adults can help determine if viability was compromised during the L2/L3 stages for further studies (the use of colored food helps with visual identification of larvae). Overall, this method can help Drosophila researchers and educators determine when viability is compromised during the fly life cycle. Routine assessment of stocks using this assay can prevent accumulation of secondary mutations that may affect the phenotype of the originally isolated mutant, especially if the original mutations affect fitness. For this reason, our lab maintains multiple copies of each of our Dm ime4 alleles and routinely checks the purity of each stock with this method in addition to other molecular analyses.&#160;

A Direct and Simple Method to Assess Drosophila melanogaster's Viability from Embryo to Adult

This protocol is designed to assess the viability of Drosophila at every developmental stage, from embryo to adult. The method can be used to determine and compare the viability of different genotypes or growth conditions.

一种直接而简单的方法，用于评估从胚胎到成人的果蝇黑色素的生存能力

In Drosophila melanogaster, viability assays are used to determine the fitness of certain genetic backgrounds. Allelic variations can result in partial or ...

Hemogenic Reprogramming of Human Fibroblasts by Enforced Expression of Transcription Factors

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human HSC emergence in vitro are required to overcome limitations in studying this complex developmental process. Here, we describe a protocol to generate hematopoietic stem and progenitor-like cells from human dermal fibroblasts employing a direct cell reprogramming approach. These cells transit through a hemogenic intermediate cell-type, resembling the endothelial-to-hematopoietic transition (EHT) characteristic of HSC specification. Fibroblasts were reprogrammed to hemogenic cells via transduction with GATA2, GFI1B and FOS transcription factors. This combination of three factors induced morphological changes, expression of hemogenic and hematopoietic markers and dynamic EHT transcriptional programs. Reprogrammed cells generate hematopoietic progeny and repopulate immunodeficient mice for three months. This protocol can be adapted towards the mechanistic dissection of the human EHT process as exemplified here by defining GATA2 targets during the early phases of reprogramming. Thus, human hemogenic reprogramming provides a simple and tractable approach to identify novel markers and regulators of human HSC emergence. In the future, faithful induction of hemogenic fate in fibroblasts may lead to the generation of patient-specific HSCs for transplantation.

This protocol demonstrates the induction of a hemogenic program in human dermal fibroblasts by enforced expression of the transcription factors GATA2, GFI1B and FOS to generate hematopoietic stem and progenitor cells.

通过转录因子的强制表达，人类纤维细胞的造血再编程

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human ...