Name: application of rnai and heat-shock-induced transcription factor expression to reprogram germ cells to neurons in c. elegans
Uploaded: 2018-01-01T15:00:00.000+00:00
Duration: 7 min 53 s
Description: Watch this Scientific Journal Video about Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans at JoVE.com

我们感谢阿琳娜 El 哈利利和纳 Hajduskova 的技术援助。我们感谢 Tursun 小组成员对手稿的评论。这项工作是由 ERC-StG-2014-637530 和 PCIG12-GA-2012-333922 的赞助, 并支持的最大 Delbrueck 中心分子医学在亥姆霍兹协会。

所有在这里描述的关于动物护理的方法已经得到德国柏林 LaGeSo 的批准。
1. 溶液制备
<ol>
	<li>NGM 琼脂板 (1 升)
	<ol>
		<li>添加3克氯化钠, 2.5 克蛋白胨培养基 (如 Bacto 蛋白胨) 和20克琼脂。灭菌后, 添加1毫升的胆固醇 (5 毫克/毫升在95% 乙醇的股票), 1 毫升 1 m MgSO4, 1 毫升1米 CaCl2, 25 毫升1米 K2PO4, 和1毫升两性霉素 B (2.5 毫克/毫升的股票)。</li>
	</ol>
	</li>
	<li>NGM 琼脂 rna 干扰板 (1 升)
	<ol>
		<li>添加3克氯化钠, 2.5 克蛋白胨培养基, 和20克琼脂。灭菌添加后, 1 毫升的胆固醇 (5 毫克/毫升在95% 乙醇的股票), 1 毫升的1米 MgSO4, 1 毫升1米 CaCl2, 25 毫升 1 m K2PO4, 1 毫升两性霉素 B (2.5 毫克/毫升库存), 1 毫升1米 IPTG和1毫升羧 (50 毫克/毫升)。</li>
	</ol>
	</li>
	<li>LB 琼脂 (1 升)
	<ol>
		<li>添加10克蛋白胨培养基, 5 克酵母提取物, 5 克氯化钠, 20 克琼脂, 10 毫升1米, pH 8.0。将 H2O 添加到 1 L。灭菌后, 加入1毫升的50毫克/毫升羧和2.5 毫升的5毫克/毫升四环素。</li>
	</ol>
	</li>
	<li>LB 培养基 (1 升)
	<ol>
		<li>添加10克蛋白胨培养基, 5 克酵母提取物, 5 克氯化钠, 10 毫升1米的 pH 8.0。将 H2O 添加到 1 L。灭菌后, 加入1毫升的50毫克/毫升羧。</li>
	</ol>
	</li>
	<li>M9-buffer (1 升)
	<ol>
		<li>添加6.0 克 Na2HPO4, 3 g,2PO4, 5 克氯化钠, 和50毫克的明胶。使用前, 添加 1 mL 1 M MgSO4。</li>
	</ol>
	</li>
	<li>漂白液 (10 毫升)
	<ol>
		<li>加入1毫升的次氯酸和2毫升的 5 N 氢氧化钠。将 H2O 添加到10毫升。</li>
	</ol>
	</li>
</ol>
2. rna 干扰板的制备
注意: 在实验室中, 线虫通常是在 6 cm 培养皿中养殖, 其中含有7.5 毫升的线虫生长培养基琼脂 (NGM 琼脂) 10,11。这个协议是优化的蠕虫保持在15° c。要用 NGM, 用标准的无菌技术来预防真菌和细菌的污染。以下是准备 NGM 板的协议, 并辅以用于 rna 干扰的试剂。
<ol>
	<li>准备6厘米 NGM 琼脂 rna 干扰板, 含有1毫米异丙基β-d--1-thiogalactopyranoside (IPTG) 和50µg/毫升羧。让他们干24–48小时在室温下在黑暗的12。
	<ol>
		<li>在黑暗中保持4° c 的 rna 干扰板。如果超过14天, 请勿使用。</li>
	</ol>
	</li>
	<li>选择带有lin-53基因 DNA 序列的 rna 干扰细菌 (大肠杆菌 HT115) 克隆, 从冷冻甘油库存中提取含有50µg/毫升的 LB 琼脂13的 L4440 质粒。羧和12.5 µg/毫升四环素使用三阶段裸奔模式。在37° c 夜间生长细菌12。此克隆允许 IPTG 依赖于细菌生产的lin-53 dsRNA。
	<ol>
		<li>此外, 增加含有 L4440 质粒的 rna 干扰细菌, 没有任何基因序列作为空矢量控制14。</li>
	</ol>
	</li>
	<li>第二天, 从lin-53或空向量 LB 琼脂板中选取一个单一的菌落, 使用200µL 吸管尖端, 并将它们接种到单独的培养基中, 其中含有2毫升液体 lb 培养基14补充50µg/ml 羧。在37° c 的夜间生长培养, 直到达到光学密度 (OD) 在600毫微米0.6 –0.8。使用分光光度计15测量 OD。 注意: 液体 lb 介质不应含有四环素, 与用于从甘油中裸奔细菌的 lb 琼脂板相反, 因为在喂养过程中加入四环素会导致 rna 干扰效率降低12</li>
	<li>添加500µL 的每一个细菌培养 (lin-53或空向量) 到6厘米 NGM 琼脂 rna 干扰板使用 multipipette。在常温下, 在室温下, 在夜间用密闭的盖子孵育盘子, 使其干燥。在这段时间内, NGM 板上的 IPTG 会诱导细菌产生 dsRNA。
	<ol>
		<li>每个实验使用至少3板每细菌培养。这将为每个实验提供3技术复制。</li>
	</ol>
	</li>
	<li>在黑暗中贮存干燥的 NGM-琼脂 rna 干扰板, 细菌在4° c, 长达两周。</li>
</ol>
3. BAT28 菌株的制备 ( C. )
<ol>
	<li>维护包含转基因otIs305的蠕虫病毒 BAT288 [hsp-16.2prom:: che-1, 6 (su1006)] 和ntIs1 [gcy-5prom::gfp] OP50 细菌使用标准 NGM 琼脂板在15° c。 注意: 有关线虫培养的详细协议, 请参见10。卷6 (su1006)是主要的等位基因和常用的表型注入标记16 , 导致典型的滚动移动。它用于otIs305转基因以跟踪hsp-16.2prom:: che-1。<ol>
		<li>保持 BAT28 应变在15° c 时刻, 以防止防范活化的hsp-16.2prom:: che-1转基因。尽量减少暴露在15° c 以上的温度, 同时处理应变。</li>
	</ol>
	</li>
	<li>要老化同步蠕虫, 请使用漂白技术17。
	<ol>
		<li>用900µL M9 缓冲液冲洗6厘米 NGM-琼脂, 含有成虫和卵 BAT28。颗粒蠕虫的离心在 900 x g, 1 分钟去除上清液。</li>
		<li>加入0.5 –1毫升的漂白溶液, 摇动管约1分钟, 直到成年蠕虫开始爆裂开放。使用标准显微镜进行监视。</li>
		<li>颗粒蠕虫的离心在 900 x g, 1 分钟去除上清液。</li>
		<li>通过在 900 x g 上加入800µL M9 缓冲液和离心, 洗涤蠕虫颗粒3次。</li>
		<li>将清洁的卵放在新鲜的 NGM 上, 用 OP50 细菌播种。在15° c 的 OP50 细菌上生长一个漂白的种群, 直到它们到达 L4 阶段 (大约4天)。L4 幼虫可以通过一个白色的补丁, 大约一半沿腹侧的蠕虫18使用标准的显微镜。 注意: 要在耗尽lin-53时实现生殖细胞到神经元转化表型, 需要在父代 (P0) 中耗尽。</li></ol></li></ol>转换表型的评分是在以下世代 (孝顺世代 F1) 进行的。为了达到消耗的lin-53已经在 P0, L4 动物受到 rna 干扰。
	
	
	<li>手动转移 50 L4 阶段蠕虫每复制使用白金线的 NGM 板, 不包含任何细菌, 让蠕虫离开任何转移 OP50 细菌。让虫子在盘子上移动大约5分钟。使用 NGM-琼脂 rna 干扰板上的细菌, 以前种子与lin-53 rna 处理细菌 (或空的矢量控制) 转移到各自的反干扰板块。
	<ol>
		<li>避免将 OP50 细菌转移到实际的 rna 干扰板上。工作快, 以尽量减少暴露时间的应变到温度高于15° c。</li>
	</ol>
	</li>
	<li>在15° c 的 NGM 琼脂 rna 干扰板上孵育蠕虫大约7天, 直到蠕虫的 F1 后代到达 L3-L4 阶段。它们可以清楚地与 P0 的动物分离, 因为它们比 F1 的后代更大更厚。
	<ol>
		<li>目视检查下标准显微镜是否 F1 子代蠕虫显示突出的外阴 (pvul) 表型, 如图 1所示。对lin-53的 rna 干扰具有效的效果, 并导致pvul表型, 可用于评估对lin-53的 rna 干扰是否成功。 注意: 对lin-53的 rna 干扰也可能导致 F1 胚胎的致命性。在动物到达 L3-L4 阶段之前, 如果板块暴露在高于15° c 的程度, 这种效应就会增加。死的胚胎可以通过被拘捕的发展和缺乏孵化认出。</li>
		<li>在黑暗中孵育板, 因为 IPTG 是轻敏感。</li>
	</ol>
	</li>

4. 诱导生殖细胞重新编程
<ol>
	<li>孵化检查含有lin-53的 rna 干扰板和空向量 rna 干扰处理的 F1 后代在黑暗中的30分钟, 在37° c 激活 CHE-1。使用一个通风箱, 因为它允许更有效的热休克。</li>
	<li>允许热休克动物在黑暗中的室温下恢复30分钟, 然后在黑暗中一夜之间在25° c 孵育盘子。</li>
	<li>使用标准的显微镜耦合到荧光光源, 检查动物的 GFP 过滤器下的gcy-5prom::gfp衍生的信号在 mid-body 区域的蠕虫, 如图 2所示。设置显微镜为 GFP 信号: 激发最大在488毫微米以发射最大在509毫微米。</li>
	<li>在琼脂填充显微镜下, 用 GFP 信号在 mid-body 区内安装动物, 评估生殖细胞与神经元转换的程度, 幻灯片19。
	<ol>
		<li>统计显示生殖细胞到神经元转化的动物数量与黑暗动物的数目, 以评估表型显。通常, 大约30% 的动物在系的lin-53耗尽时显示离散的 gfp 信号, 而不超过5% 的动物在空的矢量干扰中显示系中弥漫的 gfp 信号。</li>
	</ol>
	</li>
</ol>

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作者已经宣布, 不存在任何相互竞争的利益。

虽然所描述的协议使用转基因的线虫应变 BAT28 和 rna 干扰对lin-53是直接的, 一些步骤是关键的, 以确保生殖细胞的预期结果的神经元重新编程。重要的是, 应变 BAT28 保持在15° c 在任何时候, 在实验之前。在应变的处理和维护过程中, 温度在15° c 以上的时间需要尽可能的减少。适当的热休克条件是重要的, 因为不充分诱导的che-1过度表达不会导致生殖细胞到 ASE 神经元转换。这可以通过测量表型显, 如协议所述。广泛的热休克可能导致致命的动物。例如, 放置在内壁附近或位于静态空气孵化箱底部的板块通常会受到广泛的热处理。因此, 建议使用通风保温箱。此外, 热休克感应的时间是至关重要的, 因为表达的che-1在幼体阶段早期, 然后 L3 可能导致异位gcy-5prom::gfp诱导在不同的身体区域, 如外阴 (图 3)9. 此外, 空矢量干扰动物的表型显可检测到广泛的热休克, 不应延长 5%, 增加荧光其他组织, 即肠道。
偶尔地, rna 干扰细菌可能失去他们的活动有效地产生 dsRNA 的目标基因。不幸的是, 这在 rna 干扰实验之前不能被发现。在这种情况下, 应按照《议定书》2节的说明, 增加甘油的新的条纹。如果仍然没有 rna 干扰导致的效表型可见如pvul表型由于成功的 rna 干扰对lin-53, 然后一个质粒 DNA 分离从各自的细菌应该执行和新鲜的 HT115 细菌需要是与包含lin-53序列的 L4400 质粒进行转换。
重要的是, BAT28 应变有一个滚筒表型由注射标记 pRF4, 这是在转基因期间使用的动物与hsp-16.2prom:: che-1 DNA 结构。有时, 动物失去滚动表型, 这可能表明沉默的hsp-16.2prom:: che-1转基因。为了正确地保持 BAT28 的应变, 选择10滚子动物到一个新的板块和传播选择滚动动物。
该协议的应用限于 F1 的 rna 干扰程序, 这意味着, 其父母 (P0 代) 尚未接触到lin-53 rna 干扰的动物将不会显示生殖细胞到神经元转换, 因为产妇抢救4。Ciosk 和同事15使用了gld-1和mex-3而不表达转录因子的 rna 干扰的方法来描述将生殖细胞转换为神经元的另一种方法。然而, 获得的神经元不属于特定类型的神经元, 生殖细胞也转换成肌肉样细胞后, rna 介导的损耗gld-1和mex-315。以前, 它已经证明了对lin-53的 rna 干扰在 Pitx 型 homoedomain 转录因子 UNC-3016而不是 CHE-14的表达上允许生殖细胞转化为 gaba 神经元样细胞。.对于未来的方向, 可以测试不同的命运诱导转录因子是否可以转换为其他细胞类型比特定的神经元的lin-53衰竭生殖细胞。在这种情况下, 过度表达的肌 bHLH 转录因子 HLH-1, 同源哺乳动物 MyoD17, 诱导生殖细胞转化为肌肉在lin-53 RNAi5。在lin-53 rna 干扰和表达适当的命运诱导转录因子的基础上, 建立生殖细胞到特定体细胞类型的重新编程可以用于许多不同的遗传屏幕。这种抑制器或增强剂屏幕可以帮助解剖在细胞命运转换过程中发挥作用的调控通路。

细胞命运重新编程的异位 tf 的表达, 如肌源 tf MyoD, 当表达, 将成纤维细胞直接转化成肌肉样单元1, 几十年来一直是研究的焦点。然而, 由于保护细胞特性的机制, 分化的细胞往往不容易在这一过程中进行。生殖细胞表现出类似的保护程度, 并且有潜在的机制, 它们经常作为屏障, 防止生殖细胞转化成其他细胞类型后, 表达的命运诱导 TF。通常, 表型修饰和染色质结构等表观遗传调节会阻碍细胞命运的转换2,3。我们以前使用过的反向遗传学在线虫中进行 rna 干扰, 并确定了保护细胞特征的因素, 从而抵消了将生殖细胞重新编程为神经元的4。具体来说, polycomb 压制复合体 2 (PRC2) 和高度保守的组蛋白伴侣 LIN-53 (CAF-1/RBBP/4/7 在人类) 防止生殖细胞直接转换成特定的体细胞类型, 如谷氨酸味觉神经元在 C. 线虫4,5. 为了识别这些生殖细胞重编程障碍因子, 我们采用了携带热休克诱导型锌指 TF CHE-1 的转基因动物。CHE-1 通常表现在两个头神经元的C. 线虫, 它指定的命运谷氨酸味觉神经元称为 ASE6。ase 神经元的命运可以通过表达的 ase 特定的荧光记者gcy-5prom::gfp来可视化。GCY-5 是仅在 ASE 右神经元7中表达的化学。在lin-53中, 通过热休克干扰和诱导 CHE-1 的广泛异位表达, 生殖细胞可以转换成神经元在体内4,5。重要的是, rna 干扰处理的时间是至关重要的, 因为只有成年蠕虫 (P0 代) 暴露在lin-53 rna, 使 F1 后代动物的系, 是允许重新编程到神经元4。
上面描述的生殖细胞到神经元转换过程中的线虫可以用来研究各种生物学问题, 如信号通路在细胞命运重编程中的意义。例如, 在对lin-53进行 rna 干扰时, 我们使用 CHE-1-induced 生殖细胞重新编程为 ASE 神经元, 以研究在生殖细胞重编程过程中的缺口信号通路的含义8。通过对 co-deplete lin-53和组蛋白甲基编码基因utx-1进行双 rna 干扰, 我们表明, 在 PRC2-mediated8 中, 通过激活 UTX-1 表达式来抵消系基因沉默的缺口信号通路.这一发现表明, 生殖细胞转化为本议定书描述的神经元可以用来研究一个复杂的生物过程, 如细胞重新编程在一个完整的有机体, 并在不同的发展和环境的背景下条件.此外, 它提供的观点, 挑战生殖细胞的发酵不同的命运诱导 TFs 研究其在限制细胞可塑性的作用9, 或评估其潜在的诱导细胞重新编程的生殖细胞其他单元格类型在体内。
虽然哺乳动物的组织培养也允许研究细胞命运重新编程的不同方面, 但在文化中生长的细胞与完整的生物体相比, 模拟信号通路条件的能力有限。因此, C. 线虫是一个多用途的模型, 用于检查各种生物过程的作用, 如在重新编程体内时的切口信号。此外, 它是一个强大的基因屏幕模型, 是相对容易维护和遗传修改低成本。

<table><tbody><tr><td>&lt;strong&gt;Chemicals&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Agar-Agar, Kobe I</td><td>Roth</td><td>5210.1</td><td></td></tr><tr><td>Bactopeptone&amp;nbsp;</td><td>A. Hartenstein GmbH</td><td>211 677</td><td>Peptone Media&amp;nbsp;</td></tr><tr><td>Yeast extract&amp;nbsp;</td><td>AppliChem</td><td>A1552,0100</td><td></td></tr><tr><td>Amphotericin B</td><td>USBiological</td><td>A2220</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Merck Biosciences</td><td>208290</td><td></td></tr><tr><td>Cholesterol</td><td>Roth</td><td>8866.2</td><td></td></tr><tr><td>Gelatine</td><td>Roth</td><td>4275.3</td><td></td></tr><tr><td>IPTG</td><td>Sigma</td><td>15502-10G</td><td></td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Roth</td><td>T875.2</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Roth&amp;nbsp;</td><td>3904.2</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>VWR</td><td>25,163,364</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Roth</td><td>P030.1</td><td></td></tr><tr><td>NaCl</td><td>Roth</td><td>9265.1</td><td></td></tr><tr><td>NaClO</td><td>Roth</td><td>9062.3</td><td></td></tr><tr><td>NaOH (5 N)</td><td>Roth&amp;nbsp;</td><td>KK71.1</td><td></td></tr><tr><td>Tris</td><td>Roth</td><td>AE15.2</td><td></td></tr><tr><td>Carbenicillin</td><td>Roth</td><td>634412</td><td></td></tr><tr><td>Tetracycline</td><td>Roth</td><td>2371.2</td><td></td></tr><tr><td>&lt;strong&gt;Incubators&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Incubator&amp;nbsp;</td><td>New Brunswick Scientific C24</td><td>M1247-0052</td><td>for heat-shock</td></tr><tr><td>Incubator&amp;nbsp;</td><td>Sanyo MIR-5</td><td>5534210</td><td>for maintenance of worm strains at 15 &amp;deg;C or 25 &amp;deg;C</td></tr><tr><td>&lt;strong&gt;Microscopes&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Fluorescence microscope</td><td>M205 FA</td><td>Leica</td><td></td></tr><tr><td>Microscope (Imaging) + camera</td><td>Axio Imager 2</td><td>Zeiss</td><td></td></tr><tr><td>Microscope (Imaging) + camera</td><td>Sensicam</td><td>PCO</td><td></td></tr><tr><td>Stereomicroscope</td><td>SMZ745</td><td>Nikon</td><td></td></tr><tr><td>&lt;strong&gt;Bacterial strains&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;Escherichia coli HT115&lt;/em&gt;</td><td></td><td></td><td>F-, mcrA, mcrB, IN(rrnD-rrnE)1, rnc14::Tn10(DE3 lysogen: lavUV5&lt;br/&gt; promoter -T7 polymerase) (IPTG-inducible T7 polymerase) (RNAse&lt;br/&gt; III minus).</td></tr><tr><td>&lt;em&gt;Escherichia coli OP50&amp;nbsp;&lt;/em&gt;</td><td></td><td></td><td>Uracil auxotroph E. coli strain</td></tr><tr><td>&lt;strong&gt;Worm strains&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;BAT28:&lt;/strong&gt; &lt;em&gt;otIs305 (hsp16.2prom::che-1::&lt;br/&gt; 3xHA) ntIs1 (gcy-5prom::gfp) V.&lt;/em&gt;</td><td></td><td></td><td>derived from OH9846 by 4x times more back crossing with N2</td></tr><tr><td>&lt;strong&gt;RNAi Clones&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>lin-53</td><td>SourceBioscience</td><td>ID I-4D14</td><td>Ahringer library 16B07 ChromI, K07A1.12</td></tr><tr><td>empty vetor: L4440</td><td>Addgene&amp;nbsp;</td><td>#1654</td><td></td></tr><tr><td>&lt;strong&gt;Misc.&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Worm pick&amp;nbsp;</td><td></td><td></td><td>self-made using a pasteur pipette and a platinum wire</td></tr></tbody></table>

application of rnai and heat-shock-induced transcription factor expression to reprogram germ cells to neurons in c. elegans

研究细胞生物学过程在转换特定的细胞类型的身份, 提供了重要的洞察机制, 维护和保护细胞的身份。将生殖细胞转化为线虫的特定神经元线虫线虫 (C. 线虫) 是一种功能强大的工具, 用于执行遗传屏幕, 以便解剖保护已建立的细胞特性的调控通路。重新编程的生殖细胞, 以特定类型的神经元称为 ASE 需要转基因动物, 允许广泛表达的锌指转录因子 (TF) CHE-1。内源性 CHE-1 完全表达于两个头神经元, 并被要求指定谷氨酸 ASE 神经元的命运, 这可以很容易地被gcy-5prom::gfp的记者形象化。含有热休克启动子驱动的che-1基因表达结构的反式基因允许在热休克治疗的整个动物中广泛言不及义 CHE-1。rna 干扰与染色质调节因子 LIN-53 和热休克诱导的che-1表达的结合, 导致生殖细胞重组为 ASE 神经元样细胞。本文介绍了转基因动物热休克处理的具体的 rna 干扰程序和适宜的条件, 以成功诱导生殖细胞转化为神经元。

如图 1所示, 展示pvul表型的 F1 动物说明了lin-53 rna 干扰的成功应用。在che-1表达的热休克诱导下, 这些动物易于将生殖细胞转化为类似神经元的细胞, 如图 2所示。相反, 在控制的蠕虫, 增长的空矢量 rna 干扰lin-53 rna 干扰处理将产生的动物显示异位荧光蛋白-信号在 mid-body 地区后热休克和25° c 孵化过夜如前所述, 4,8. 在这些蠕虫的荧光显微镜下进行更仔细的检查可以发现, 显示 GFP 信号的细胞也会显示神经元样的投影 (图 2B, 白色箭头), 这是神经元细胞的典型特征。如果对lin-53的 rna 干扰不成功或热休克条件不合适, GFP 信号将类似于控制 rna 干扰处理的动物 (图 2)。重要的是, 在动物到达中 L3 阶段之前, 避免诱发表达的热诱导che-1 。太年轻的动物在che-1过度表达诱导时, 可以在身体的其他区域显示异位 gcy-5prom::gfp , 如图 313中所示的开发外阴。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56889/56889fig1.jpg"> 
图 1:对 lin-53 的 rna 干扰引起的 Pvul 表型.顶)L4/young 成虫期 F1 子代的 DIC 图片从控件派生的蠕虫或lin-53 rna 干扰处理的母亲。缩放条形图 = 20 µm. (底部) 动物用lin-53 rna 处理显示突出的外阴 (pvul) 表型 (黑色箭头头)。pvul表型确认对lin-53的 rna 干扰是成功的。缩放栏 = 20 µm.<a href="//ecsource.jove.com/files/ftp_upload/56889/56889fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56889/56889fig2.jpg"> 
图 2:蠕虫将生殖细胞成功地重新编程成神经元.(A) BAT28 在空向量上生长的应变动物, 在系热休克诱导后, 不显示 GFP 信号, 在che-1过度表达。虚线空心线轮廓蠕虫。缩放条形图 = 20 µm. (B) 与 BAT28 应变动物生长在空矢量控制 rna 干扰动物生长在lin-53 rna 干扰显示gcy-5prom::gfp信号在 mid-body 地区。mid-body 部分的图片与 gfp 信号采取在63X 放大显示 gfp 阳性细胞显示突起 (白色箭头头)。这种形态学特征表明生殖细胞经转化为类似于神经元样细胞。白色星号标记肠衍生荧光。所有的照片都是使用荧光显微镜, 20X 放大倍率和63X 放大。虚线白线轮廓蠕虫。缩放栏 = 20 µm.<a href="//ecsource.jove.com/files/ftp_upload/56889/56889fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56889/56889fig3.jpg"> 
图 3:具有异位荧光蛋白的早期蠕虫.BAT28 应变动物生长的空矢量 rna 干扰细菌, 是热休克诱发的che-1过度表达在早期幼体阶段, 如 L2 显示 GFP 信号在 mid-body 地区 (白星号)。这些信号不是由于生殖细胞重新编程。虚线空心线轮廓蠕虫。缩放栏 = 20 µm.<a href="//ecsource.jove.com/files/ftp_upload/56889/56889fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans at JoVE.com

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

rna 干扰和热休克诱导转录因子表达在重编程生殖细胞到神经元中的应用C. 线虫

本协议描述了如何在细胞命运转换期间研究在线虫线虫体内的细胞过程。利用转基因动物, 允许热休克启动子驱动的神经元的过度表达, 诱导转录因子 CHE-1 和 rna 介导的染色质调节因子 LIN-53 生殖细胞对神经元重新编程的损耗可以观察在体内。

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain ...

application-rnai-heat-shock-induced-transcription-factor-expression

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

7.6K 次观看.  Max Delbrück Center for Molecular Medicine in the Helmholtz Association. 本协议描述了如何在细胞命运转换期间研究在线虫线虫体内的细胞过程。利用转基因动物, 允许热休克启动子驱动的神经元的过度表达, 诱导转录因子 CHE-1 和 rna 介导的染色质调节因子 LIN-53 生殖细胞对神经元重新编程的损耗可以观察在体内。 

视频: Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Standardized Methods for Measuring Induction of the Heat Shock Response in Caenorhabditis elegans

The heat shock response (HSR) is a cellular stress response induced by cytosolic protein misfolding that functions to restore protein folding homeostasis, or proteostasis. Caenorhabditis elegans occupies a unique and powerful niche for HSR research because the HSR can be assessed at the molecular, cellular, and organismal levels. Therefore, changes at the molecular level can be visualized at the cellular level and their impacts on physiology can be quantitated at the organismal level. While assays for measuring the HSR are straightforward, variations in the timing, temperature, and methodology described in the literature make it challenging to compare results across studies. Furthermore, these issues act as a barrier for anyone seeking to incorporate HSR analysis into their research. Here, a series of protocols is&#160;presented for measuring induction of the HSR in a robust and reproducible manner with RT-qPCR, fluorescent reporters, and an organismal thermorecovery assay. Additionally, we show that a widely used thermotolerance assay is not dependent on the well-established master regulator of the HSR, HSF-1, and therefore should not be used for HSR research. Finally, variations in these assays found in the literature are discussed and best practices are proposed to help standardize results across the field, ultimately facilitating neurodegenerative disease, aging, and HSR research.

Standardized Methods for Measuring Induction of the Heat Shock Response in Caenorhabditis elegans

Here, standardized protocols are presented to assess induction of the heat shock response (HSR) in Caenorhabditis elegans using RT-qPCR at the molecular level, fluorescent reporters at the cellular level, and thermorecovery at the organismal level.

卡诺哈布迪蒂斯精英热休克反应的测量诱导标准化方法

The heat shock response (HSR) is a cellular stress response induced by cytosolic protein misfolding that functions to restore protein folding homeostasis, ...

科研

环境科学

秀丽隐杆线虫的表观遗传遗传和 ChIP

Analysis of Transgenerational Epigenetic Inheritance in C. elegans Using a Fluorescent Reporter and Chromatin Immunoprecipitation (ChIP)

Transgenerational epigenetic inheritance (TEI) allows the transmission of information through the germline without changing the genome sequence, through factors such as non-coding RNAs and chromatin modifications. The phenomenon of RNA interference (RNAi) inheritance in the nematode Caenorhabditis elegans is an effective model to investigate TEI that takes advantage of this model organism's short life cycle, self-propagation, and transparency. In RNAi inheritance, exposure of animals to RNAi leads to gene silencing and altered chromatin signatures at the target locus that persist for multiple generations in the absence of the initial trigger. This protocol describes the analysis of RNAi inheritance in C. elegans using a germline-expressed nuclear green fluorescent protein (GFP) reporter. Reporter silencing is initiated by feeding the animals bacteria expressing double-stranded RNA targeting GFP. At each generation, animals are passaged to maintain synchronized development, and reporter gene silencing is determined by microscopy. At select generations, populations are collected and processed for chromatin immunoprecipitation (ChIP)-quantitative polymerase chain reaction (qPCR) to measure histone modification enrichment at the GFP reporter locus. This protocol for studying RNAi inheritance can be easily modified and combined with other analyses to further investigate TEI factors in small RNA and chromatin pathways.

作者聚焦:秀丽隐杆线虫的 RNAi 遗传和 ChIP 

This protocol describes an RNA interference and ChIP assay to study the epigenetic inheritance of RNAi-induced silencing and associated chromatin modifications in C. elegans.

基于荧光报告基因和染色质免疫沉淀法（ChIP）分析 秀丽隐杆线虫 的跨代表观遗传

Transgenerational epigenetic inheritance (TEI) allows the transmission of information through the germline without changing the genome sequence, through ...

遗传学

Osmotic Avoidance in Caenorhabditis elegans: Synaptic Function of Two Genes, Orthologues of Human NRXN1 and NLGN1, as Candidates for Autism

Neurexins and neuroligins are cell adhesion molecules present in excitatory and inhibitory synapses, and they are required for correct neuron network function1. These proteins are found at the presynaptic and postsynaptic membranes 2. Studies in mice indicate that neurexins and neurologins have an essential role in synaptic transmission 1. Recent reports have shown that altered neuronal connections during the development of the human nervous system could constitute the basis of the etiology of numerous cases of autism spectrum disorders 3.
Caenorhabditis elegans could be used as an experimental tool to facilitate the study of the functioning of synaptic components, because of its simplicity for laboratory experimentation, and given that its nervous system and synaptic wiring has been fully characterized. In C. elegans nrx-1 and nlg-1 genes are orthologous to human NRXN1 and NLGN1 genes which encode alpha-neurexin-1 and neuroligin-1 proteins, respectively. In humans and nematodes, the organization of neurexins and neuroligins is similar in respect to functional domains.
The head of the nematode contains the amphid, a sensory organ of the nematode, which mediates responses to different stimuli, including osmotic strength. The amphid is made of 12 sensory bipolar neurons with ciliated dendrites and one presynaptic terminal axon 4. Two of these neurons, named ASHR and ASHL are particularly important in osmotic sensory function, detecting water-soluble repellents with high osmotic strength 5. The dendrites of these two neurons lengthen to the tip of the mouth and the axons extend to the nerve ring, where they make synaptic connections with other neurons determining the behavioral response 6.
To evaluate the implications of neurexin and neuroligin in high osmotic strength avoidance, we show the different response of C. elegans mutants defective in nrx-1 and nlg-1 genes, using a method based on a 4M fructose ring 7. The behavioral phenotypes were confirmed using specific RNAi clones 8. In C. elegans, the dsRNA required to trigger RNAi can be administered by feeding 9. The delivery of dsRNA through food induces the RNAi interference of the gene of interest thus allowing the identification of genetic components and network pathways.

Osmotic Avoidance in Caenorhabditis elegans: Synaptic Function of Two Genes, Orthologues of Human NRXN1 and NLGN1, as Candidates for Autism

Neurexins and neuroligins are membrane-neuron adhesion proteins which perform essential roles in synaptic differentiation and transmission. Neuroligin deficient mutants of C. elegans are defective in detecting osmotic strength, but when they also contain a mutation in the gene coding neurexin, they recover the wild type phenotype.

渗透避免IN线虫：突触功能的两个基因，人类同源 NRXN1 NLGN1，作为候选人自闭症

Neurexins and neuroligins are cell adhesion molecules present in excitatory and inhibitory synapses, and they are required for correct neuron network ...

生物学

Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

This protocol describes the use of fluorescence microscopy to image dividing cells within developing Caenorhabditis elegans embryos. In particular, this protocol focuses on how to image dividing neuroblasts, which are found underneath the epidermal cells and may be important for epidermal morphogenesis. Tissue formation is crucial for metazoan development&#160;and relies on external cues from neighboring tissues. C. elegans is an excellent model organism to study tissue morphogenesis in vivo due to its transparency and simple organization, making its tissues easy to study via microscopy. Ventral enclosure is the process where the ventral surface of the embryo is covered by a single layer of epithelial cells. This event is thought to be facilitated by the underlying neuroblasts, which provide chemical guidance cues to mediate migration of the overlying epithelial cells. However, the neuroblasts are highly proliferative and also may act as a mechanical substrate for the ventral epidermal cells. Studies using this experimental protocol could uncover the importance of intercellular communication during tissue formation, and could be used to reveal the roles of genes involved in cell division within developing tissues.

Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

This protocol describes how to image dividing cells within a tissue in Caenorhabditis elegans embryos. While several protocols describe how to image cell division in the early embryo, this protocol describes how to image cell division within a developing tissue during mid late embryogenesis.

可视化的神经细胞胞质分裂过程中<em&gt; C。线虫</em&gt;胚胎

This protocol describes the use of fluorescence microscopy to image dividing cells within developing Caenorhabditis elegans embryos. In particular, this ...

神经科学

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

RNAi筛选识别胚后的表型 C。线虫</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. elegans, gene function can be knocked down simply and efficiently by feeding worms with bacteria expressing a dsRNA corresponding to a specific gene 1. While the creation of libraries of RNAi clones covering most of the C. elegans genome 2,3 opened the way for true functional genomic studies (see for example 4-7), most established methods are laborious. Moy and colleagues have developed semi-automated protocols that facilitate genome-wide screens 8. The approach relies on microscopic imaging and image analysis. 
	Here we describe an alternative protocol for a high-throughput genome-wide screen, based on robotic handling of bacterial RNAi clones, quantitative analysis using the COPAS Biosort (Union Biometrica (UBI)), and an integrated software: the MBioLIMS (Laboratory Information Management System from Modul-Bio) a technology that provides increased throughput for data management and sample tracking. The method allows screens to be conducted on solid medium plates. This is particularly important for some studies, such as those addressing host-pathogen interactions in C. elegans, since certain microbes do not efficiently infect worms in liquid culture.
	We show how the method can be used to quantify the importance of genes in anti-fungal innate immunity in C. elegans. In this case, the approach relies on the use of a transgenic strain carrying an epidermal infection-inducible fluorescent reporter gene, with GFP under the control of the promoter of the antimicrobial peptide gene nlp 29 and a red fluorescent reporter that is expressed constitutively in the epidermis. The latter provides an internal control for the functional integrity of the epidermis and nonspecific transgene silencing9. When control worms are infected by the fungus they fluoresce green. Knocking down by RNAi a gene required for nlp 29 expression results in diminished fluorescence after infection. Currently, this protocol allows more than 3,000 RNAi clones to be tested and analyzed per week, opening the possibility of screening the entire genome in less than 2 months.

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

We describe a protocol using C. elegans and RNAi feeding libraries that allows automated measurement of multiple parameters such as fluorescence, size and opacity of individual worms in a population. We give one example of a screen to identify genes involved in anti-fungal innate immunity in C. elegans.

定量和自动化高通量的全基因组RNAi屏幕 C。线虫</em

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. ...

Tissue-Specific RNAi Tools to Identify Components for Systemic Stress Signaling

Over the past decade there has been a transformative increase in knowledge surrounding the regulation of protein quality control processes, unveiling the importance of intercellular signaling processes in the regulation of cell-nonautonomous proteostasis. Recent studies are now beginning to uncover signaling components and pathways that coordinate protein quality control from one tissue to another. It is therefore important to identify mechanisms and components of the cell-nonautonomous proteostasis network (PN) and its relevance for aging, stress responses and protein misfolding diseases. In the laboratory, we use genetic knockdown by tissue-specific RNAi in combination with stress reporters and tissue-specific proteostasis sensors to study this. We describe methodologies to examine and to identify components of the cell-nonautonomous PN that can act in tissues perceiving a stress condition and in responding cells to activate a protective response. We first describe how to generate hairpin RNAi constructs for constitutive genetic knockdown in specific tissues and how to perform tissue-specific genetic knockdown by feeding RNAi at different life stages. Stress reporters&#160;and behavioral assays function as&#160;valuable readouts&#160;that enable&#160;the fast screening of genes and conditions modifying systemic stress signaling processes. Finally, proteostasis sensors expressed in different tissues are utilized to determine changes in the tissue-specific capacity of the PN at different stages of development and aging. Thus, these tools should help clarify and allow monitoring the capacity of PN in specific tissues, while helping to identify components that function in different tissues to mediate cell-nonautonomous PN in an organism.

Maintenance of organismal proteostasis requires the coordination of protein quality control responses such as chaperone expression from one tissue to another. Here, we provide tools used in C. elegans that allow monitoring of proteostasis capacity in specific tissues and determine intercellular signaling responses.

Over the past decade there has been a transformative increase in knowledge surrounding the regulation of protein quality control processes, unveiling the ...

RNAi in C. elegans

RNA interference (RNAi) is a widely used technique in which double stranded RNA is exogenously introduced into an organism, causing knockdown of a target gene. In the nematode, C. elegans, RNAi is particularly easy and effective because it can be delivered simply by feeding the worms bacteria that express double stranded RNA (dsRNA) that is complementary to a gene of interest. First, this video will introduce the concept of RNA interference and explain how it causes targeted gene knockdown. Then, we will demonstrate a protocol for using RNAi in C. elegans, which includes preparation of the bacteria and RNAi worm plates, culturing of the worms, and how to assess the effects of RNAi on the worms. RNAi is frequently used to perform reverse genetic screens in order to reveal which genes are important to carry out specific biological processes. Furthermore, automated reverse genetic screens allow for the efficient knockdown and analysis of a large collection of genes. Lastly, RNAi is often used to study the development of C. elegans. Since its discovery, scientists have used RNAi to make tremendous progress on the understanding of many biological phenomena.

RNA Interference RNAi in Caenorhabditis elegans

线虫RNA干扰的简介

RNA interference (RNAi) is a widely used technique in which double stranded RNA is exogenously introduced into an organism, causing knockdown of a target ...

rna 干扰和热休克诱导转录因子表达在重编程生殖细胞到神经元中的应用C. 线虫

摘要

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基于荧光报告基因和染色质免疫沉淀法（ChIP）分析秀丽隐杆线虫的跨代表观遗传

渗透避免IN线虫：突触功能的两个基因，人类同源 NRXN1 NLGN1，作为候选人自闭症

可视化的神经细胞胞质分裂过程中

RNAi筛选识别胚后的表型 C。线虫

定量和自动化高通量的全基因组RNAi屏幕 C。线虫

Tissue-Specific RNAi Tools to Identify Components for Systemic Stress Signaling

线虫RNA干扰的简介

rna 干扰和热休克诱导转录因子表达在重编程生殖细胞到神经元中的应用C. 线虫

摘要

探索更多视频

此视频中的章节

卡诺哈布迪蒂斯精英热休克反应的测量诱导标准化方法

基于荧光报告基因和染色质免疫沉淀法（ChIP）分析 秀丽隐杆线虫 的跨代表观遗传

基于荧光报告基因和染色质免疫沉淀法（ChIP）分析 秀丽隐杆线虫 的跨代表观遗传

基于荧光报告基因和染色质免疫沉淀法（ChIP）分析 秀丽隐杆线虫 的跨代表观遗传

渗透避免IN线虫：突触功能的两个基因，人类同源 NRXN1 NLGN1，作为候选人自闭症

可视化的神经细胞胞质分裂过程中

RNAi筛选识别胚后的表型 C。线虫

定量和自动化高通量的全基因组RNAi屏幕 C。线虫

Tissue-Specific RNAi Tools to Identify Components for Systemic Stress Signaling

线虫RNA干扰的简介

基于荧光报告基因和染色质免疫沉淀法（ChIP）分析秀丽隐杆线虫的跨代表观遗传

基于荧光报告基因和染色质免疫沉淀法（ChIP）分析秀丽隐杆线虫的跨代表观遗传

基于荧光报告基因和染色质免疫沉淀法（ChIP）分析秀丽隐杆线虫的跨代表观遗传