作者宣布没有利益冲突。

总的协议需要2天, 包括第一天的败血症诱导和分离从动物的球液, 第二天的外来体隔离从老鼠 BALF。所有程序都已由亚特兰大 VA 医疗中心的机构动物护理和使用委员会审查和批准。
1. 小鼠急性脓毒性肺损伤模型
<ol><li>使用 6-8 周-老雄性野生型 C57BL/6J 小鼠 (重量 20-22 克) 的动物模型, 并执行所有程序使用无菌技术和仪器。高压釜所有手术器械, 包括镊子和剪刀, 20 分钟121摄氏度。</li>
	<li>将小鼠随机分布到实验和对照组。在手术板上抑制老鼠。用腹腔途径治疗小鼠与大肠杆菌脂多糖 (LPS, 15 毫克/千克). 根据个人体重, 稀释适当的 lps 剂量为每只老鼠0.1 毫升 PBS, 然后注射 lps 溶液使用1毫升注射器与27克针和注射一个同等数量的 PBS 控制动物。</li>
	<li>保持老鼠在动物 BSL2 设施, 其中包括干净的笼子在适当的室温。</li>
	<li>24小时后, 弄死小鼠与 CO2吸入。</li>
	<li>选择鼠标颈部中心的手术部位。做一个小切口 (大约5毫米), 并轻轻地分开肌肉, 使用钝钳进入气管, 直到气管暴露。</li>
	<li>用27克针提取10毫升无菌注射器的小鼠球液。首先, 轻轻地, 插入针入气管和注射1毫升的不育 PBS 慢慢进入气管, 然后绘制 BALF 成一个3毫升一次性注射器与27克针。程序完成后, 在生化利器容器中处理注射器和针头。</li>
	<li>用二氧化氯治疗外科器械, 如前所述的 Chauret et 。27, 然后用热处理20分钟在121摄氏度消毒所有手术器械。</li>
</ol>2. 外来体隔离-串联超离心和表征
<ol><li>收集15毫升锥形管中的小鼠球液。为了收割肺泡细胞, 离心机样品10分钟在1000克, 在4摄氏度。</li>
	<li>将上清到新的15毫升圆锥管, 然后离心样品30分钟在 4000 x g 在4°c 去除细胞碎片。</li>
	<li>收集在 ultracentrifuge 管和离心样品60分钟在 1万 x g 4 摄氏度的上清, 以消除较大的细胞颗粒。</li>
	<li>将上清液转移到新的锥形管, 然后通过0.2 µm 注射器过滤器过滤样品, 以除去剩余的较大颗粒。</li>
	<li>收集 ultracentrifuge 管和平衡管的样品。离心样品2小时, 在 14万 x g 在 4°c, 以颗粒的外来体。 注: 外来体的大小变化为 20-200 nm。0.2 µm 过滤器用于去除大小超过200纳米的较大粒子。</li>
	<li>轻轻丢弃上清液。用100µL PBS 或溶解溶液溶解颗粒, 并将外来体样品转移到1.5 毫升管, 并贮存在-80 摄氏度直至使用。 注: 建议使用透射电镜验证隔离外来体。</li>
	<li>按照标准 TEM 示例处理协议28。用透射电镜分析外来体样品, 并拍摄图像, 缩放条 200 nm。</li>
</ol>3. 制备 miRNA 和执行 miRNA-q-rt-pcr
注: miRNA-q-rt-pcr 是分析 miRNAs 表达水平的建议。
<ol><li>遵循标准的 miRNAs 隔离协议, 并确保在单链反向转录中使用等量的纯化 rna24。</li>
	<li>按照标准的实时 PCR 协议检测 exosomal microRNA 水平。使用特定的实时 PCR 引物对 miR-155, miR-146a 和 U6 SnRNA。用定量 pcr 仪进行 pcr 反应, 将表达值规范化为内部控制 U6 snRNA。 注: 每个组由三个样本24组成。</li>
</ol>4. 外来体标记和纯化
<ol><li>悬浮外来体颗粒与100µL 悬浮液在1.5 毫升聚丙烯管, 然后添加0.4 µL 的外来体染料进入另一管100µL 的悬液。</li>
	<li>快速添加外来体溶液中的染料溶液, 并将其彻底混合吹打。在室温下保持样品5分钟, 免受光照。</li>
	<li>在1.5 毫升洗脱管中放入一个外切旋转柱。将解决方案混合物转移到外切旋转列的中心。</li>
	<li>在室温下, 在 800 x g 处旋转柱2分钟。</li>
	<li>丢弃该列并将洗脱样本存储在-20 摄氏度, 直到使用。</li>
</ol>5. 接受方细胞外来体的验证
<ol><li>种子 1 x 104在8井室幻灯片中的 12/单元格, 以及在37°c、5% CO2上的培养单元。</li>
	<li>在细胞中添加10µL 荧光标记的外来体。在37摄氏度孵育1小时。</li>
	<li>去除培养基, 用 PBS 冲洗细胞, 室温下固定1% 多聚甲醛的细胞10分钟。</li>
	<li>用 DAPI 装入装入介质中的幻灯片。</li>
	<li>通过荧光显微镜分析样品, 拍摄图像, 放大40X。</li>
</ol>6. 验证外来体与接收细胞之间的相互作用
<ol><li>种子 2 x 105 MLE12/well 在包含适当培养基的12井培养板中, 在37°c、5% CO2上培养细胞。</li>
	<li>将20µg 外来体从小鼠的体液中添加到 MLE12 细胞中, 并建立每个实验组的复制。</li>
	<li>24小时后, 用 PBS 冲洗细胞, 并将600µL 溶解缓冲剂放入井中。轻轻摇动盘子, 在1.5 毫升管内用吸管收集裂解样品。</li>
	<li>遵循标准的总 RNA 隔离协议和 cDNA 反向转录协议, 完成单链 cDNA 反向转录29。</li>
	<li>按照标准实时 PCR 协议检测收件人单元格中的 mRNA 表达式级别29。使用特定的实时 PCR 引物抗肿瘤坏死因子α, IL-6 和 ZO-1。用定量 pcr 仪进行 pcr 反应, 将表达值规范化为内部控制 GAPDH。 注: 每组由三份样品组成。</li>
</ol>

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这项工作得到 VA 价值审查2I01BX001786-06A1 资助 RS 的支持。

小鼠疾病模型通常用于评估特定基因的生理功能, 并降低实验费用2。这里描述的急性脓毒性肺损伤模仿人与 ARDS 的炎症反应。该模型与研究分子发病机制、生物标志物的发展以及测试潜在的新疗法5有关。
共培养系统与体内研究有关, 它是在对体外系统23、30中的生物分子 (如 RNA、DNA、脂质?...

阿里和 ARDS 是一种危及生命的呼吸衰竭, 其严重的低氧血症由非心源性肺水肿引起, 每年全球约有100万人感染1。急性呼吸窘迫综合征的病因包括感染或吸入对肺部的直接损伤和各种间接侮辱。在过去的十年中, 人们对 ards 的分子发病机制有了越来越多的了解, 但是, 针对 ards 的特定靶向治疗还有待开发2,3。
建立了几种急性肺损伤动物模型, 为人类研究的实验治疗提供了一座桥梁4,5。通常使用的模型包括油酸、细菌、LPS 和博莱霉素的本地安装。其他方法包括缺血再灌注、盲肠结扎穿刺、机械通气引起的伸展损伤、高氧或系统性细菌的管理和 LPS5。这些模型提供了一个有用的生物学系统来测试临床假说和发展潜在的治疗方法。为了模拟人 ARDS, 动物模型应重现炎症和急性损伤的上皮和内皮细胞的缺陷, 在屏障功能的肺部。
外来体是 20-200 nm 直径的膜泡, 其分子含量包含蛋白质、DNA、RNA 和脂质, 并通过分子组成转移促进组织微环境中的细胞间通信。外来体由多种类型的细胞分泌, 如内皮细胞、上皮细胞、平滑肌细胞和肿瘤细胞, 并存在于人体体液中。研究表明, 外来体调节免疫细胞和基质细胞在传染性和无菌炎症性疾病中的交叉交谈, 其异常释放似乎受到各种自然和实验性刺激的调节, 在生理和病理过程6。这种通信网络可能在肺部疾病的发病机制中发挥重要作用, 并可能影响病理生理学进展7,8。作为 18-22 核苷酸非编码 rna, miRNAs 存在于组织和体液, 血浆, 血清和调制 mRNA 表达在后平移水平9,10。
包装 miRNAs 在外来体影响分化和多种类型细胞的功能, 过多的水平是与各种疾病, 包括癌症, 肺部疾病, 肥胖, 糖尿病和心血管疾病, 11, 12,13,14,15,16。进入接受细胞和穿梭 exosomal miRNAs 促进细胞间通讯, 改变微环境的止血17,18。急性肺损伤是一个复杂的过程, 涉及多细胞类型与广泛的细胞间通讯通过外来体 8.miR-155 和 miR-146a 共享共同的转录调节机制, 并有助于炎症反应和免疫耐受性19,20。最近的研究表明, 两种调节炎症反应通过 exosomal miRNAs 穿梭在免疫细胞之间的21。然而, exosomal miRNAs 对内毒素的肺泡反应的调节作用的分子机制尚不清楚, 毫无疑问, 潜在的临床相关性和转化暗示值得进一步研究。
共用培养模型用于定义复杂环境中特定细胞类型的相互作用, 如炎症和癌症22,23。这些平台提供了一个替代的策略, 以询问细胞类型之间的交叉谈话, 特别是免疫和结构细胞。
内毒素的气管内、雾化、腹腔或全身管理被广泛用于诱发实验性肺损伤24,25,26, 并显示诱导上皮和内皮渗透性缺陷。在这里我们用腹腔内毒素诱导小鼠急性肺损伤的脓毒性模型。在内毒素腹腔内24小时内, 肺部出现通透性缺损, 并招募炎症细胞。此外, 我们还表明, 外来体从 miRNA-155 和 miR-146a, 和外来体从球液诱导促炎细胞因子表达的受体上皮细胞, 包括 IL-6 和 TNF α。这些数据是首次表明, exosomal miRNAs 是分泌的急性脓毒症肺损伤模型。

<table border="0" cellpadding="0" cellspacing="0" style="width:1276px;" width="1276">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:691px;">lipopolysaccharide</td>
			<td style="width:181px;">Sigma-Aldrich</td>
			<td style="width:224px;">Escherichia coli 055:B5</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS pH7.2(1X)</td>
			<td>Life technologies</td>
			<td>20012-027</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">mirVana miRNA isolation kit</td>
			<td>Thermo Fisher</td>
			<td align="right">449774</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TaqMan Gene Expression Master Mix</td>
			<td>Thermo Fisher</td>
			<td align="right">4369016</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">mmu-miR-155 (002571) primer</td>
			<td>Thermo Fisher</td>
			<td align="right">4427975</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">has-miR-146a (000468) primer</td>
			<td>Thermo Fisher</td>
			<td align="right">4427975</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">U6snRNA primer</td>
			<td>Thermo Fisher</td>
			<td align="right">4427975</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TNF-&#945;(Mm00443258_m1 ) primer</td>
			<td>Thermo Fisher</td>
			<td align="right">4331182</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IL-6 (Mm00446190_m1&#160;&#160; )primer</td>
			<td>Thermo Fisher</td>
			<td align="right">4331182</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ZO-1 (Mm00493699_m1&#160; )primer</td>
			<td>Thermo Fisher</td>
			<td align="right">4331182</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">minimal essential medium (MEM)</td>
			<td>Thermo Fisher</td>
			<td>11095-072</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trysin-EDTA solution(0.05%)</td>
			<td>Thermo Fisher</td>
			<td align="right">25300054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Exosomes were labeled with PKH67 through PKH67 Green Fluorescent Cell linker Mini Kit&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>MINI67-1KT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Exosome Spin Columns (MW3000)</td>
			<td>Thermo Fisher</td>
			<td align="right">4484449</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Beckman Coulter Optima L-100XP ultracentrifuge&#160;</td>
			<td>Beckman Coulter</td>
			<td>L-100XP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultra-clear centrifuge tubes</td>
			<td>Beckman Coulter</td>
			<td align="right">344058</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Applied Biosystems 7500 Fast Real-Time PCR System</td>
			<td>Thermo Fisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">transmission electron microscopes (TEM)</td>
			<td>JEOL Ltd</td>
			<td>120 kV TEM</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Olympus BX41 microscope</td>
			<td>Olympus</td>
			<td>BX41</td>
			<td></td>
		</tr>
	</tbody>
</table>

bronchoalveolar lavage exosomes in lipopolysaccharide-induced septic lung injury

急性肺损伤 (ALI) 和急性呼吸窘迫综合征 (ARDS) 是一种不同种类的肺部疾病, 持续高发病率和死亡率。阿里的分子发病机制得到了更好的界定;然而, 由于疾病的复杂性质, 分子疗法还有待开发。在这里, 我们使用脂多糖 (LPS) 诱导小鼠急性脓毒性肺损伤模型, 描绘了外来体在炎症反应中的作用。使用这个模型, 我们可以表明, 暴露于腹腔内 LPS 分泌外来体的小鼠从肺部的支气管肺泡灌洗 (球) 液中, 包裹着 miRNA 和细胞因子, 调节炎症反应。进一步使用共培养模型系统, 我们表明, 外来体释放的巨噬细胞扰乱紧张连接蛋白的表达在支气管上皮干细胞。这些结果表明, 1) 通过 exosomal 穿梭的先天免疫和结构细胞之间的交叉交谈, 有助于炎症反应和破坏的结构屏障和 2) 针对这些 miRNAs 可能提供一个新的平台, 以治疗阿里和 ARDS。

为诱发脓毒性肺损伤, 小鼠采用腹腔内 LPS (15 毫克/千克) 治疗。在 LPS 管理的24小时内, 中性的涌入出现在肺部, 如图 1A所示。绘制了小鼠球液, 然后分离纯化了外来体。用电子透射显微镜 (图 1B) 证实了 BALF 外来体的形态学。
从纯化外来体中检测 miRNA-155 和 miR-146a 的选择性促炎 miRNAs ?...

Watch this Scientific Journal Video about Bronchoalveolar Lavage Exosomes in Lipopolysaccharide-induced Septic Lung Injury at JoVE.com

Bronchoalveolar Lavage Exosomes in Lipopolysaccharide-induced Septic Lung Injury

支气管肺泡灌洗外来体在脂多糖致脓毒性肺损伤中的作用

暴露于腹腔内的 LPS 分泌外来体的小鼠支气管肺泡灌洗 (球) 液, 包装与 miRNAs。使用共培养系统, 我们表明, 外来体释放的球液扰乱支气管上皮细胞紧密连接蛋白的表达, 并增加支持炎症细胞因子的表达, 加重肺损伤。

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) represent a heterogeneous group of lung diseases which continues to have a high ...

bronchoalveolar-lavage-exosomes-lipopolysaccharide-induced-septic

Research

JoVE Journal

Genetics

10.8K 次观看.  Atlanta VAMC. 暴露于腹腔内的 LPS 分泌外来体的小鼠支气管肺泡灌洗 (球) 液, 包装与 miRNAs。使用共培养系统, 我们表明, 外来体释放的球液扰乱支气管上皮细胞紧密连接蛋白的表达, 并增加支持炎症细胞因子的表达, 加重肺损伤。

视频: Bronchoalveolar Lavage Exosomes in Lipopolysaccharide-induced Septic Lung Injury

siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend diseases related to pregnancy. In this protocol, human primary villous cytotrophoblasts freshly isolated from placentas through a standard DNase/trypsin protocol are microporated with small interfering RNA (siRNA). This approach provided greater efficiency for siRNA transfection when compared to a lipofection-based method. Transfected cells can subsequently be analyzed by standard Western blot within a time frame of 3-4 days post-transfection. In addition, using cultured primary villous cytotrophoblasts, Electrophoretic Mobility Shift Assay (EMSA) analysis was optimized and performed on extracts from days 1 to 4. The use of these cultured primary cells and the protocol described allow for an evaluation of the implication of specific genes and transcription factors in the process of villous cytotrophoblast differentiation into a syncytiotrophoblast-like cell layer. However, the limited time span allowable in culture precludes the use of methods requiring more time, such as generation of a stable cell population. Therefore testing of this cell population requires highly optimized gene transfer protocols.

This protocol describes a method for efficiently transfecting siRNA in freshly isolated human villous cytotrophoblasts using microporation and identifying DNA-protein complexes in these cells. Transfected cells can be monitored by Western blot and EMSA analyses during the 4-day culture time.

在新鲜分离的人代绒毛滋养细胞转染和EMSA分析

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend ...

科研

遗传学

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and transmitted from one generation to the next through the female oocyte is of fundamental importance. Because of the genetic tractability, and the elegant, ordered simplicity by which oocyte development proceeds, Drosophila oogenesis has become an invaluable system for mitochondrial study. An EdU (5-ethynyl-2&#180;-deoxyuridine) labeling method was utilized to detect mitochondrial DNA (mtDNA) replication in Drosophila ovaries. This method is superior to the BrdU (5-bromo-2'-deoxyuridine) labeling method in that it allows for good structural preservation and efficient fluorescent dye penetration of whole-mount tissues.
Here we describe a detailed protocol for labeling replicating mitochondrial DNA in Drosophila adult ovaries with EdU. Some technical solutions are offered to improve the viability of the ovaries, maintain their health during preparation, and ensure high-quality imaging. Visualization of newly synthesized mtDNA in the ovaries not only reveals the striking temporal and spatial pattern of mtDNA replication through oogenesis, but also allows for simple quantification of mtDNA replication under various genetic and pharmacological perturbations.

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

Drosophila oogenesis continues to be exceptionally useful in the study of mitochondrial proliferation and inheritance. This manuscript describes a detailed protocol used to label the replicating mitochondrial DNA (mtDNA) in Drosophila adult ovaries with 5-ethynyl-2&#180;-deoxyuridine (EdU), which facilitates uncovering mechanisms associated with mitochondrial inheritance that were previously debatable.

在 果蝇卵巢成人线粒体DNA复制的原位标记埃杜染色

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and ...

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most mutagenesis-based screens, researchers have relied on the use of toxic chemicals, carcinogens, or irradiation, which requires designated equipment, safety setup, and/or disposal of hazardous materials. We have developed a simple approach to induce heritable mutations in C. elegans using germline-expressed histone-miniSOG, a light-inducible potent generator of reactive oxygen species. This mutagenesis method is free of toxic chemicals and requires minimal laboratory safety and waste management. The induced DNA modifications include single-nucleotide changes and small deletions, and complement those caused by classical chemical mutagenesis. This methodology can also be used to induce integration of extrachromosomal transgenes. Here, we provide the details of the LED setup and protocols for standard mutagenesis and transgene integration.

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Genetically-encoded histone-miniSOG induces genome-wide heritable mutations in a blue&#160;light-dependent manner. This mutagenesis method is simple, fast, free of toxic chemicals, and well-suited for forward genetic screening and transgene integration.

光遗传学随机突变用组蛋白miniSOG在<em&gt;℃。线虫</em

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most ...

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative without tools to study gene function. The development and sharing of detailed protocols for the establishment of new model systems in laboratories, and for tools to carry out functional studies, is thus crucial for leveraging the power of genomics. Coleoptera (beetles) are the largest clade of insects and occupy virtually all types of habitats on the planet. In addition to providing ideal models for fundamental research, studies of beetles can have impacts on pest control as they are often pests of households, agriculture, and food industries. Detailed protocols for rearing and maintenance of D. maculatus laboratory colonies and for carrying out dsRNA-mediated interference in D. maculatus are presented. Both embryonic and parental RNAi procedures-including apparatus set up, preparation, injection, and post-injection recovery-are described. Methods are also presented for analyzing embryonic phenotypes, including viability, patterning defects in hatched larvae, and cuticle preparations for unhatched larvae. These assays, together with in situ hybridization and immunostaining for molecular markers, make D. maculatus an accessible model system for basic and applied research. They further provide useful information for establishing procedures in other emerging insect model systems.

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Here, we present protocols for rearing an intermediate-germ beetle, Dermestes maculatus (D. maculatus) in the lab. We also share protocols for embryonic and parental RNAi and methods for analyzing embryonic phenotypes to study gene function in this species.

饲养并在隐藏甲壳虫双链RNA介导的基因敲除，<em&gt;白腹皮蠹</em

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative ...

Pooled shRNA Screen for Reactivation of MeCP2 on the Inactive X Chromosome

Forward genetic screens using reporter genes inserted into the heterochromatin have been extensively used to investigate mechanisms of epigenetic control in model organisms. Technologies including short hairpin RNAs (shRNAs) and clustered regularly interspaced short palindromic repeats (CRISPR) have enabled such screens in diploid mammalian cells. Here we describe a large-scale shRNA screen for regulators of X-chromosome inactivation (XCI), using a murine cell line with firefly luciferase and hygromycin resistance genes knocked in at the C-terminus of the methyl CpG binding protein 2 (MeCP2) gene on the inactive X-chromosome (Xi). Reactivation of the construct in the reporter cell line conferred survival advantage under hygromycin B selection, enabling us to screen a large shRNA library and identify hairpins that reactivated the reporter by measuring their post-selection enrichment using next-generation sequencing. The enriched hairpins were then individually validated by testing their ability to activate the luciferase reporter on Xi.

We report a small hairpin RNA (shRNA) and next generation sequencing-based protocol for identifying regulators of X-chromosome inactivation in a murine cell line with firefly luciferase and hygromycin resistance genes fused to the methyl CpG binding protein 2 (MeCP2) gene on the inactive X chromosome.

在非活动 X 染色体上重新激活 MeCP2 的池 shRNA 屏幕

Forward genetic screens using reporter genes inserted into the heterochromatin have been extensively used to investigate mechanisms of epigenetic control ...

Formaldehyde-assisted Isolation of Regulatory Elements to Measure Chromatin Accessibility in Mammalian Cells

Appropriate gene expression in response to extracellular cues, that is, tissue- and lineage-specific gene transcription, critically depends on highly defined states of chromatin organization. The dynamic architecture of the nucleus is controlled by multiple mechanisms and shapes the transcriptional output programs. It is, therefore, important to determine locus-specific chromatin accessibility in a reliable fashion that is preferably independent from antibodies, which can be a potentially confounding source of experimental variability. Chromatin accessibility can be measured by various methods, including the Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) assay, that allow the determination of general chromatin accessibility in a relatively low number of cells. Here we describe a FAIRE protocol that allows simple, reliable, and fast identification of genomic regions with a low protein occupancy. In this method, the DNA is covalently bound to the chromatin proteins using formaldehyde as a crosslinking agent and sheared to small pieces. The free DNA is afterwards enriched using phenol:chloroform extraction. The ratio of free DNA is determined by quantitative polymerase chain reaction (qPCR) or DNA sequencing (DNA-seq) compared to a control sample representing total DNA. The regions with a looser chromatin structure are enriched in the free DNA sample, thus allowing the identification of genomic regions with lower chromatin compaction.

The plasticity of nuclear architecture is controlled by dynamic epigenetic mechanisms including non-coding RNAs, DNA methylation, nucleosome repositioning as well as histone composition and modification. Here we describe a Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) protocol which allows the determination of chromatin accessibility in a reproducible, inexpensive, and straightforward manner.

甲醛辅助分离测定哺乳动物细胞染色质可及性的调控元素

Appropriate gene expression in response to extracellular cues, that is, tissue- and lineage-specific gene transcription, critically depends on highly ...

CRISPR-Mediated Reorganization of Chromatin Loop Structure

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene expression, but whether looping is responsible for or a result of alterations in gene expression is still unknown. Until recently, how chromatin looping affects the regulation of gene activity and cellular function has been relatively ambiguous, and limitations in existing methods to manipulate these structures prevented in-depth exploration of these interactions. To resolve this uncertainty, we engineered a method for selective and reversible chromatin loop re-organization using CRISPR-dCas9 (CLOuD9). The dynamism of the CLOuD9 system has been demonstrated by successful localization of CLOuD9 constructs to target genomic loci to modulate local chromatin conformation. Importantly, the ability to reverse the induced contact and restore the endogenous chromatin conformation has also been confirmed. Modulation of gene expression with this method establishes the capacity to regulate cellular gene expression and underscores the great potential for applications of this technology in creating stable de novo chromatin loops that markedly affect gene expression in the contexts of cancer and development.

Chromatin looping plays a significant role in gene regulation; however, there have been no technological advances that allow for selective and reversible modification of chromatin loops. Here we describe a powerful system for chromatin loop re-organization using CRISPR-dCas9 (CLOuD9), demonstrated to selectively and reversibly modulate gene expression at targeted loci.

CRISPR 的染色质环结构重组

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene ...

Identification of Homologous Recombination Events in Mouse Embryonic Stem Cells Using Southern Blotting and Polymerase Chain Reaction

Relative to the issues of off-target effects and the difficulty of inserting a long DNA fragment in the application of designer nucleases for genome editing, embryonic stem (ES) cell-based gene-targeting technology does not have these shortcomings and is widely used to modify animal/mouse genome ranging from large deletions/insertions to single nucleotide substitutions. Notably, identifying the relatively few homologous recombination (HR) events necessary to obtain desired ES clones is a key step, which demands accurate and reliable methods. Southern blotting and/or conventional PCR are often utilized for this purpose. Here, we describe the detailed procedures of using those two methods to identify HR events that occurred in mouse ES cells in which the endogenous Myh9 gene is intended to be disrupted and replaced by cDNAs encoding other nonmuscle myosin heavy chain IIs (NMHC IIs). The whole procedure of Southern blotting includes the construction of targeting vector(s), electroporation, drug selection, the expansion and storage of ES cells/clones, the preparation, digestion, and blotting of genomic DNA (gDNA), the hybridization and washing of probe(s), and a final step of autoradiography on the X-ray films. PCR can be performed directly with prepared and diluted gDNA. To obtain ideal results, the probes and restriction enzyme (RE) cutting sites for Southern blotting and the primers for PCR should be carefully planned. Though the execution of Southern blotting is time-consuming and labor-intensive and PCR results have false positives, the correct identification by Southern blotting and the rapid screening by PCR allow the sole or combined application of these methods described in this paper to be widely used and consulted by most labs in the identification of genotypes of ES cells and genetically modified animals.

Here, we present a detailed protocol for identifying homologous recombination events that occurred in mouse embryonic stem cells using Southern blotting and/or PCR. This method is exemplified by the generation of nonmuscle myosin II genetic replacement mouse models using traditional embryonic stem cell-based homologous recombination-mediated targeting technology.

利用南方印迹和聚合酶链反应鉴定小鼠胚胎干细胞的同源重组事件

Relative to the issues of off-target effects and the difficulty of inserting a long DNA fragment in the application of designer nucleases for genome ...

Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives each year. Effective treatment options able to halt disease progression are lacking. Despite the extensive sequencing efforts in large patient populations, the majority of ALS and PD cases remain unexplained by genetic mutations alone. Epigenetics mechanisms, such as the post-translational modification of histone proteins, may be involved in neurodegenerative disease etiology and progression and lead to new targets for pharmaceutical intervention. Mammalian in vivo and in vitro models of ALS and PD are costly and often require prolonged and laborious experimental protocols. Here, we outline a practical, fast, and cost-effective approach to determining genome-wide alterations in histone modification levels using Saccharomyces cerevisiae as a model system. This protocol allows for comprehensive investigations into epigenetic changes connected to neurodegenerative proteinopathies that corroborate previous findings in different model systems while significantly expanding our knowledge of the neurodegenerative disease epigenome.

This protocol outlines experimental procedures to characterize genome-wide changes in the levels of histone post-translational modifications (PTM) occurring in connection with the overexpression of proteins associated with ALS and Parkinson&#39;s disease in Saccharomyces cerevisiae models. After SDS-PAGE separation, individual histone PTM levels are detected with modification-specific antibodies via Western blotting.

酵母神经退行性蛋白病模型中组蛋白后转化修饰改变的表征

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives ...

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant viruses they vector. Despite numerous studies of the biology of these species in different environments, a key life history parameter, offspring sex ratios, has received little attention, yet is important for predicting population dynamics. The primary sex ratio (sex ratio at oviposition) of Bemisia tabaci has never been reported but can be found by determining the egg fertilization rate of this haplodiploid insect. The technique involves the dechorionation of eggs with bleach, a series of fixation steps, and the application of the general DNA fluorescent stain, DAPI (4&#8242;,6-diamidino-2-phenylindole, a DNA-binding fluorescent dye), to bind to female and male pronuclei. Here, we present the technique, and an example of its application, to test whether an endosymbiotic bacterium, Rickettsia sp. nr. bellii, influenced the primary sex ratio of B. tabaci. This method may assist in population studies of whiteflies, or in determining if sex allocation exists with certain environmental stimuli.

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

We present a simple cytogenetic technique using 4&#8242;,6-diamidino-2-phenylindole (DAPI) to determine the fertilization rate and primary sex ratio of the haplodiploid invasive pest Bemisia tabaci.

用细胞遗传学技术测定芽孢杆菌的受精率

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant ...

支气管肺泡灌洗外来体在脂多糖致脓毒性肺损伤中的作用

摘要

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此视频中的章节

在新鲜分离的人代绒毛滋养细胞转染和EMSA分析

在果蝇卵巢成人线粒体DNA复制的原位标记埃杜染色

光遗传学随机突变用组蛋白miniSOG在

饲养并在隐藏甲壳虫双链RNA介导的基因敲除，

在非活动 X 染色体上重新激活 MeCP2 的池 shRNA 屏幕

甲醛辅助分离测定哺乳动物细胞染色质可及性的调控元素

CRISPR 的染色质环结构重组

利用南方印迹和聚合酶链反应鉴定小鼠胚胎干细胞的同源重组事件

酵母神经退行性蛋白病模型中组蛋白后转化修饰改变的表征

用细胞遗传学技术测定芽孢杆菌的受精率

支气管肺泡灌洗外来体在脂多糖致脓毒性肺损伤中的作用

摘要

探索更多视频

此视频中的章节

在新鲜分离的人代绒毛滋养细胞转染和EMSA分析

在 果蝇卵巢成人线粒体DNA复制的原位标记埃杜染色

光遗传学随机突变用组蛋白miniSOG在

饲养并在隐藏甲壳虫双链RNA介导的基因敲除，

在非活动 X 染色体上重新激活 MeCP2 的池 shRNA 屏幕

甲醛辅助分离测定哺乳动物细胞染色质可及性的调控元素

CRISPR 的染色质环结构重组

利用南方印迹和聚合酶链反应鉴定小鼠胚胎干细胞的同源重组事件

酵母神经退行性蛋白病模型中组蛋白后转化修饰改变的表征

用细胞遗传学技术测定芽孢杆菌的受精率

在果蝇卵巢成人线粒体DNA复制的原位标记埃杜染色