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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Chemicals&#160;</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&#160;</td>
			<td>A. Hartenstein GmbH</td>
			<td>211 677</td>
			<td>Peptone Media&#160;</td>
		</tr>
		<tr>
			<td>Yeast extract&#160;</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>CaCl2</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>K2PO4</td>
			<td>Roth</td>
			<td>T875.2</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Roth&#160;</td>
			<td>3904.2</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>VWR</td>
			<td>25,163,364</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</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&#160;</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>Incubators</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator&#160;</td>
			<td>New Brunswick Scientific C24</td>
			<td>M1247-0052</td>
			<td>for heat-shock</td>
		</tr>
		<tr>
			<td>Incubator&#160;</td>
			<td>Sanyo MIR-5</td>
			<td>5534210</td>
			<td>for maintenance of worm strains at 15 &#176;C or 25 &#176;C</td>
		</tr>
		<tr>
			<td>Microscopes</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>Bacterial strains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Escherichia coli HT115</td>
			<td></td>
			<td></td>
			<td>F-, mcrA, mcrB, IN(rrnD-rrnE)1, rnc14::Tn10(DE3 lysogen: lavUV5 
			promoter -T7 polymerase) (IPTG-inducible T7 polymerase) (RNAse 
			III minus).</td>
		</tr>
		<tr>
			<td>Escherichia coli OP50&#160;</td>
			<td></td>
			<td></td>
			<td>Uracil auxotroph E. coli strain</td>
		</tr>
		<tr>
			<td>Worm strains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BAT28: otIs305 (hsp16.2prom::che-1:: 
			3xHA) ntIs1 (gcy-5prom::gfp) V.</td>
			<td></td>
			<td></td>
			<td>derived from OH9846 by 4x times more back crossing with N2</td>
		</tr>
		<tr>
			<td>RNAi Clones</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&#160;</td>
			<td>#1654</td>
			<td></td>
		</tr>
		<tr>
			<td>Misc.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Worm pick&#160;</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

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

Application of Impermeable Barriers Combined with Candidate Factor Soaked Beads to Study Inductive Signals in the Chick

The chick embryo provides a superb vertebrate model that can be used to dissect developmental questions in a direct way. Its accessibility and robustness following surgical intervention are key experimental strengths. Mica plates were the first barriers used to prevent chick limb bud initiation1. Protocols that use aluminum foil as an impermeable barrier to wing bud or leg bud induction and or initiation are described. We combine this technique with bead placement lateral to the barrier to exogenously supply candidate endogenous factors that have been blocked by the barrier. The results are analyzed using in situ hybridization of subsequent gene expression. Our main focus is on the role of retinoic acid signaling in the induction and later initiation of the chick embryo fore and hindlimb. We use BMS 493 (an inverse agonist of retinoic acid receptors (RAR)) soaked beads implanted in the lateral plate mesoderm (LPM) to mimic the effect of a barrier placed between the somites (a source of retinoic acid (RA)) and the LPM from which limb buds grow. Modified versions of these protocols could also be used to address other questions on the origin and timing of inductive cues. Provided the region of the chick embryo is accessible at the relevant developmental stage, a barrier could be placed between the two tissues and consequent changes in development studied. Examples may be found in the developing brain, axis extension and in organ development, such as liver or kidney induction.

Protocols using impermeable barriers to block induction events between tissues of the main body axis and flank in the chick embryo required for limb formation are described. Beads soaked in candidate inductive signals are used to overcome the effect of barrier placement and analysis of gene expression confirms this.

在小鸡学习感应信号与候选因子浸泡珠结合防渗屏障中的应用

The chick embryo provides a superb vertebrate model that can be used to dissect developmental questions in a direct way. Its accessibility and robustness ...

科研

发育生物学

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple cellular and developmental processes principally via activation of ERK, the terminal kinase of the pathway. Tight regulation of ERK activity is essential for normal development and homeostasis; overly active ERK results in excessive cellular proliferation, while underactive ERK causes cell death. C. elegans is a powerful model system that has helped characterize the function and regulation of RTK-RAS-ERK signaling pathway during development. In particular, the RTK-RAS-ERK pathway is essential for C. elegans germline development, which is the focus of this method. Using antibodies specific to the active, diphosphorylated form of ERK (dpERK), the stereotypical localization pattern can be visualized within the germline. Because this pattern is both spatially and temporally controlled, the ability to reproducibly assay dpERK is useful to identify regulators of the pathway that affect dpERK signal duration and amplitude and thus germline development. Here we demonstrate how to successfully dissect, stain, and image dpERK within the C. elegans gonad. This method can be adapted for spatial localization of any signaling or structural protein in the C. elegans gonad, provided an antibody compatible with immunofluorescence is available.

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

We present an immunofluorescence&#160;imaging-based method for spatial and temporal localization of active ERK in the dissected C. elegans gonad. The protocol described here can be adapted for visualization of any signaling or structural protein in the C. elegans gonad, provided a suitable antibody reagent is available.

在Active ERK的时空分析<em&gt;℃。线虫</em&gt;生殖系

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple ...

Assessing Cardiomyocyte Subtypes Following Transcription Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts

Direct reprogramming of one cell type into another has recently emerged as a powerful paradigm for regenerative medicine, disease modeling, and lineage specification. In particular, the conversion of fibroblasts into induced cardiomyocyte-like myocytes (iCLMs) by Gata4, Hand2, Mef2c, and Tbx5 (GHMT) represents an important avenue for generating de novo cardiac myocytes in vitro and in vivo. Recent evidence suggests that GHMT generates a greater diversity of cardiac subtypes than previously appreciated, thus underscoring the need for a systematic approach to conducting additional studies. Before direct reprogramming can be used as a therapeutic strategy, however, the mechanistic underpinnings of lineage conversion must be understood in detail to generate specific cardiac subtypes. Here we present a detailed protocol for generating iCLMs by GHMT-mediated reprogramming of mouse embryonic fibroblasts (MEFs). 
We outline methods for MEF isolation, retroviral production, and MEF infection to accomplish efficient reprogramming. To determine the subtype identity of reprogrammed cells, we detail a step-by-step approach for performing immunocytochemistry on iCLMs using a defined set of compatible antibodies. Methods for confocal microscopy, identification, and quantification of iCLMs and individual atrial (iAM), ventricular (iVM), and pacemaker (iPM) subtypes are also presented. Finally, we discuss representative results of prototypical direct reprogramming experiments and highlight important technical aspects of our protocol to ensure efficient lineage conversion. Taken together, our optimized protocol should provide a stepwise approach for investigators to conduct meaningful cardiac reprogramming experiments that require identification of individual CM subtypes.

This manuscript describes a step-by-step protocol for the generation and quantification of diverse reprogrammed cardiac subtypes using a retrovirus-mediated delivery of Gata4, Hand2, Mef2c, and Tbx5.

评估心肌亚型以下鼠标胚胎成纤维细胞的转录因子介导的重编程

Direct reprogramming of one cell type into another has recently emerged as a powerful paradigm for regenerative medicine, disease modeling, and lineage ...

A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. Currently, it allows the discrimination of up to 9 murine germ cell populations with high yield and purity. This high-resolution in discrimination and purification is possible due to unique changes in chromatin structure and quantity throughout spermatogenesis. These patterns can be captured by flow cytometry of male germ cells stained with fluorescent DNA-binding dyes such as Hoechst-33342 (Hoechst). Herein is a detailed description of a recently developed protocol to isolate mammalian testicular germ cells. Briefly, single cell suspensions are generated from testicular tissue by mechanical dissociation, double stained with Hoechst and propidium iodide (PI) and processed by flow cytometry. A serial gating strategy, including the selection of live cells (PI negative) with different DNA content (Hoechst intensity), is used during FACS sorting to discriminate up to 5 germ cell types. These include, with corresponding average purities (determined by microscopy evaluation): spermatogonia (66%), primary (71%) and secondary (85%) spermatocytes, and spermatids (90%), further separated into round (93%) and elongating (87%) subpopulations. Execution of the entire workflow is straightforward, allows the isolation of 4 cell types simultaneously with the appropriate FACS machine, and can be performed in less than 2 h. As reduced processing time is crucial to preserve the physiology of ex vivo cells, this method is ideal for downstream high-throughput studies of male germ cell biology. Moreover, a standardized protocol for multispecies purification of mammalian germ cells eliminates methodological sources of variables and allows a single set of reagents to be used for different animal models.

This work describes the standardization of a method to obtain purified germ cell populations from testicular tissue of different mammalian species. It is a straightforward protocol that combines mechanical testis dissociation, staining with Hoechst-33342 and propidium iodide, and FACS sorting, with wide applications in comparative studies of male reproductive biology.

通过机械组织分离和流式细胞术对哺乳动物雄性生殖细胞多特异性纯化的标准化方法

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. ...

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein are molecular motors that regulate anterograde and retrograde transport, respectively. These motors use microtubule networks as rails. Caenorhabditis elegans (C. elegans) is a powerful model organism to study axonal transport and IFT in vivo. Here, I describe a protocol to observe axonal transport and IFT in living C. elegans. Transported cargo can be visualized by tagging cargo proteins using fluorescent proteins such as green fluorescent protein (GFP). C. elegans is transparent and GFP-tagged cargo proteins can be expressed in specific cells under cell-specific promoters. Living worms can be fixed by microbeads on 10% agarose gel without killing or anesthetizing the worms. Under these conditions, cargo movement can be directly observed in the axons and cilia of living C. elegans without dissection. This method can be applied to the observation of any cargo molecule in any cells by modifying the target proteins and/or the cells they are expressed in. Most basic proteins such as molecular motors and adaptor proteins that are involved in axonal transport and IFT are conserved in C. elegans. Compared to other model organisms, mutants can be obtained and maintained more easily in C. elegans. Combining this method with various C. elegans mutants can clarify the molecular mechanisms of axonal transport and IFT.

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Caenorhabditis elegans (C. elegans) is a good model to study axonal and intracellular transport. Here, I describe a protocol for in vivo recording and analysis of axonal and intraflagellar transport in C. elegans.

固定化的线虫线虫分析神经元内细胞的转运

Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

秀丽线虫生殖的计算分析研究细胞核、蛋白质和细胞骨架的分布

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

人诱导多能干细胞体外生成体细胞衍生物的研究

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Hemogenic Endothelium Differentiation from Human Pluripotent Stem Cells in A Feeder- and Xeno-free Defined Condition

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous transplants to treat hematological and malignant disorders. Hemogenic endothelium (HE) is an amenable platform to produce functional HSPCs by transcription factors or to induce lymphocytes in a robust manner. However, current methods to derive HE are either costly or variable in yield. In this protocol, we established a cost-effective and precise method to derive HE within 4 days in a feeder- and xeno-free defined condition. The resultant HE underwent an endothelial-to-hematopoietic transition and produced CD34+CD45+ clonogenic hematopoietic progenitors. The potential capacity of our platform for generating functional HSPCs will have broad applications in disease modeling and screening for therapeutic compounds.

Here, we present a protocol to precisely form hemogenic endothelium from human pluripotent stem cells in a feeder- and xeno-free condition. This method will have broad applications in disease modeling and screening for therapeutic compounds.

在无进料和异种定义的条件下,从人类多能干细胞中血性内皮物分化

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous ...

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational health hazard. Here, we demonstrate the exposure of primary stem cells from the mouse ocular surface to UV-C radiation. Reactive oxygen species (ROS) formation is the readout of the extent of oxidative stress/damage. In an experimental in vitro setting, it is also essential to assess the percentage of dead cells generated due to oxidative stress. In this article, we will demonstrate the 2&#39;,7&#39;-Dichlorofluoresceindiacetate (DCFDA) staining of UV-C exposed mouse primary ocular surface stem cells and their quantification based on the fluorescent images of DCFDA staining. DCFDA staining directly corresponds to ROS generation. We also demonstrate the quantification of dead and live cells by simultaneous staining with propidium iodide (PI) and Hoechst 3332 respectively and the percentage of DCFDA (ROS positive) and PI positive cells.

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

This protocol demonstrates the simultaneous detection of reactive oxygen species (ROS), live cells, and dead cells in live primary cultures from mouse ocular surface cells. 2&#39;,7&#39;-Dichlorofluoresceindiacetate, propidium iodide, and Hoechst staining are used to assess the ROS, dead cells, and live cells, respectively, followed by imaging and analysis.

评估原小鼠眼部表面细胞/干细胞对紫外线-C （UV-C） 损伤的反应

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...

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 ...

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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此视频中的章节

在小鸡学习感应信号与候选因子浸泡珠结合防渗屏障中的应用

在Active ERK的时空分析

评估心肌亚型以下鼠标胚胎成纤维细胞的转录因子介导的重编程

通过机械组织分离和流式细胞术对哺乳动物雄性生殖细胞多特异性纯化的标准化方法

固定化的线虫线虫分析神经元内细胞的转运

秀丽线虫生殖的计算分析研究细胞核、蛋白质和细胞骨架的分布

人诱导多能干细胞体外生成体细胞衍生物的研究

在无进料和异种定义的条件下,从人类多能干细胞中血性内皮物分化

评估原小鼠眼部表面细胞/干细胞对紫外线-C （UV-C）损伤的反应

通过转录因子的强制表达，人类纤维细胞的造血再编程

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

在小鸡学习感应信号与候选因子浸泡珠结合防渗屏障中的应用

在Active ERK的时空分析

评估心肌亚型以下鼠标胚胎成纤维细胞的转录因子介导的重编程

通过机械组织分离和流式细胞术对哺乳动物雄性生殖细胞多特异性纯化的标准化方法

固定化的线虫线虫分析神经元内细胞的转运

秀丽线虫生殖的计算分析研究细胞核、蛋白质和细胞骨架的分布

人诱导多能干细胞体外生成体细胞衍生物的研究

在无进料和异种定义的条件下,从人类多能干细胞中血性内皮物分化

评估原小鼠眼部表面细胞/干细胞对紫外线-C （UV-C） 损伤的反应

通过转录因子的强制表达，人类纤维细胞的造血再编程

评估原小鼠眼部表面细胞/干细胞对紫外线-C （UV-C）损伤的反应