这项工作是在伍斯特理工学院的支持下进行的。

1. 制备线虫生长培养基 (NGM) <ol> <li> 用于 1 l 介质, 结合 20 g 琼脂、2.5 g 有机氮源 ( 如 、bacto-蛋白胨) 和3克氯化钠在 2 l 烧瓶中。加入975毫升的无菌水. 
	<ol> <li> 在无菌搅拌栏中添加。如果使用自动介质倒, 高压釜管和介质为15分钟;如果有更高的音量, 介质应更长的时间进行蒸压. </li>
	</ol> </li> <li> 在搅拌板上设置介质, 并允许冷却。
	<ol> <li> 一旦媒体被热触碰 (大约60和 #176; C) 菌增加1毫升的1米 MgSO 4 和 #9679; H 2 O, 1 毫升的 1M CaCl 2 , 25 毫升的 KPO 4 , 和1毫升的0.5% 的胆固醇在乙醇中. </li>
		<li> 将介质 (手动或自动泵) 倒入 35 mm x 10 mm 无菌培养皿中。允许在使用前1-2 天晾干引擎盖. 
		注: 板材应存储在4和 #176; C, 以防止污染. </li>
	</ol> </li> </ol> 2。制作蠕虫选择 <ol> <li> 将一根1.5 厘米的铝线切下。小心地将大约0.5 厘米的长度插入到巴斯德吸管的尖端. 
	<ol> <li> 使用本生燃烧器或酒精灯, 将玻璃吸管尖端慢慢地旋转到火焰中, 直到导线牢固地附着在吸管上, 而不会损害吸管尖端的原始形状. </li>
	</ol> </li> </ol> 3。 大肠杆菌 培养基的制备 <ol> <li> 使用无菌循环, 在200毫升的 Luria 汤中接种单一菌落的 大肠杆菌 菌株 OP50。在37和 #176 摇晃过夜; C. </li>
	<li> 液体养殖已准备就绪, 可在次日使用, 并可存储在4和 #176; C 时不使用. 
	注意: OP50 的 大肠杆菌 菌株被用作 C. 线虫的食物来源 此区域性可在4和 #176 存储时使用长达几个月; C. </li>
</ol> 4。基本的 线虫 维护 <ol> <li> 种子准备 NGM 琼脂板与 大肠杆菌 , 通过发现大约 50-100 和 #181; L 准备 OP50 液体文化到板材的中心. </li>
	<li> 盖板, 使其在室温下干燥24小时。一旦干燥, 放置在37和 #176 的盘子; C 隔夜为迅速成长或保持在室温下, 如果增长是渴望的. </li>
	<li> 通过任意和 #34 将蠕虫添加到板中; 分块、#34; (请参阅协议步骤5和 #34; 将蠕虫和 #34;), 逐个选取蠕虫, 或将蠕虫卵直接放到板上 (参见协议步骤6和 #34; 蛋准备和 #34;). </li>
</ol> 5。分块和拾取蠕虫 注意: 和 #34; #34. 是指在 C. 线虫 维护中常见的一种做法。这涉及切割含有蠕虫的 NGM 琼脂板的一部分, 并将其转移到一个新的板块, 从而也转移了大量的蠕虫在这个过程中。污染也可以用这种方式从盘子里切出来。当采摘蠕虫时, 重要的是要保持琼脂的完整性, 因为蠕虫可以在琼脂的孔中丢失。此外, 重要的是, 让采摘冷却前触摸板, 以防止融化的琼脂或燃烧的蠕虫. 
<ol> <li> 为了快速将大量的蠕虫转移到新的板上, 请用刮刀将含有所选蠕虫的一块 NGM 琼脂切开。删除切割件, 并将其面朝下的 OP50 草坪的边缘上的一个新的板块. </li>
	<li> 在协议步骤8.2、#34、生存分析和 #34 中分别传输蠕虫; 使用蠕虫拾取。在蠕虫的选择中火焰, 直到它是红色的。从火焰中取出, 让它冷却几秒钟. </li>
	<li> 用铁丝在镐上, 轻轻刮去 OP50 文化的草坪上生长的新盘子, 盖上铁丝上的镐. </li>
	<li> 在导线尖端的粘性培养物会使蠕虫粘在拾取的尖端, 从而用滑动运动轻轻地拾取选定的蠕虫. </li>
	<li> 一旦选定的蠕虫被收集, 将它们放到一个新的盘子上, 轻轻地在琼脂上滑动区域性。将导线放入火焰中, 以去除拾取中的剩余区域性. </li>
</ol> 6。鸡蛋准备 <ol> <li> 将大约20成年动物 (大约 2-3 天后孵化时, 生长在20和 #176; C) 按协议步骤 5, #34; 分块和采摘蠕虫, 和 #34; 在一个有50个种子的盘子上和 #181 大肠杆菌菌株, OP50. </li>
	<li> 收集鸡蛋, 在1-2 天后用10毫升的 M9 缓冲液淹没盘子。使用玻璃血清吸管, 旋流, 并轻轻地搅动琼脂板上的内容, 以释放鸡蛋粘在 OP50 或成年动物。收集所有含有卵和成虫的液体. </li>
	<li> 将 M9 和 OP50 溶液转换为15毫升锥形管。离心机15毫升锥管在 2100 x g 2 min. </li>
	<li> 吸气约9毫升的上清, 不干扰 OP50 颗粒. </li>
	<li> 重2毫升无菌水中的颗粒, 1 毫升的漂白剂和1毫升的0.25 米氢氧化钠. </li>
	<li> 通过反转轻轻混合, 直到成年动物的大多数 (大约 70%) 出现裂解; 通常情况下, 由于蛋壳的原因, 鸡蛋在漂白过程中保持完好。离心锥管在 990 x g 为 2 min. </li>
	<li> 吸入上清液而不干扰颗粒。重新悬浮在10毫升的 M9 缓冲颗粒. </li>
	<li> 离心锥管 990 x g, 2 分钟, 无干扰颗粒。200、#181 的 M9 缓冲颗粒重新悬浮. 
	注: 卵子准备可能需要多次试验。如果鸡蛋暴露在漂白剂中太久, 它们可能会被破坏。如果鸡蛋不孵化, 要么减少溶液中漂白剂的浓度, 要么减少接触漂白剂的时间. </li>
</ol> 7。感染板设置 <ol> <li> 将3-5 毫升的 YPD 分配到一个无菌试管中, 并使用一个无菌循环将它接种到一个单一菌落的白色念珠菌 中. 
	<ol> <li> 将导管放置在旋转鼓上大约16-18 小时, 在30和 #176; C. </li>
	</ol> </li> <li> 标记一个1.5 毫升离心管, 以包含 C. 白色念珠菌 , 另一个包含 OP50。记录每个空管的重量. 
	<ol> <li> 将500和 #181; 过夜的 c. 白色念珠菌 培养成管标为 白色念珠菌 和1500和 #181; 夜间 OP50 培养 (从步骤 3.1) 到标有 OP50 的管子. </li>
		<li> 离心10分钟, 16100 x g. 吸上清液而不干扰颗粒. </li>
		<li> 重新挂起500和 #181 的每个颗粒; 无菌水的 L。离心机为5分钟, 在 16100 x g. </li>
		<li> 吸入上清液而不干扰颗粒。记录离心管的最终重量. </li><li> 通过从最终重量中减去离心管的初始重量来确定每个颗粒的重量. </li>
	</ol> </li> <li> 使用无菌水, 将 re-suspend 为10毫克/毫升, 并将 OP50 颗粒至200毫克/毫升。通过结合10和 #181; 50 毫克/毫升链霉素, 2.5 和 #181; l 的 OP50 培养, 0.5 和 #181; l. 白色念珠菌 培养, 7 和 #181; 无菌水的 l. 
	<ol> <li> 对于控制板, 通过用无菌水替换 C. 白色念珠菌 的体积来创建主混合。种子20和 #181; L 感染混合或 OP50 控制解决方案, NGM 板的中心使用微. 
		注: 在分析过程中, 需要大约100蠕虫来确定统计意义。这种化验通常是三份的。因此, 一个3.2x 的感染混合以及3.2x 控制解决方案作出。这些混合被制成略超过3x 的原始, 以确保一个完整的20和 #181; 我被播种在每一个盘子上, 允许有错误的空间。此主组合是创建的, 因为在初始幼体阶段 (L1-L3) 中, 蠕虫的咽太小, 无法使用 C. 白念珠菌 。对于接触药物, 如氟康唑 ( 图 3B ), 该剂被引入到感染混合物。药剂的集中是经验主义地坚定的。例如, 在 图 3B 中, 50 和 #181; m 氟康唑的代表, 但 0, 12.5, 25, 50 和100和 #181; m 氟康唑也使用相同的协议进行了测试. </li>
	</ol> </li> </ol> 8。变形肛门区 (dar) 表型与生存试验分析 <ol> <li> 可视化 dar 表型 注: dar 在 L3 阶段和更远处是最好的可视化。Dar 在蠕虫的后肛门区域 ( 图 2A ) 中显示为一个小突起, 随着时间的推移, 它变得更加突出。
	<ol> <li> 通过在解剖显微镜的舞台上快速敲击板来确认动物是否有 dar; 没有 dar 的动物将立即向后移动, 而与 dar 的动物将需要反复的敲击来反转它们的方向, 或者不会这样做. </li>
	</ol> </li> <li> 生存分析 <ol> <li> 完整的蛋准备, 如协议步骤6中所述, #34; 鸡蛋准备和 #34;。计算下解剖范围内的卵, 用 M9 稀释卵子溶液, 直到每 #181 约5-6 健康卵的浓度; 我成功了使用吸管, 免除20和 #181; 样品中含有约30鸡蛋在准备的 NGM 板上, 在细菌培养和侧板之间的区域 (卵和蠕虫食物, OP50, 是在两个不同的斑点). </li>
		<li> 在20和 #176 孵育板; C 为48小时。在解剖显微镜下, 统计每个板块上的活的和死的成虫数, 以及与 Dar 的蠕虫数量。用 Dar 记录百分比. </li>
		<li> 如果它不响应由铝线敲击头部或在板上敲击, 则认为是动物死亡. </li>
		<li> 每隔一天, 按协议步骤5中所述的方法选择转移剩余的成虫, 并 #34; 分块和拾取蠕虫、#34; 进入新的感染或控制板。在这一点上, 如果需要, 通过跟踪每天的活、死和缺失蠕虫的数量来进行生存试验. 
		注: 此化验可运行两周, 从准备卵子开始。此外, 卵子的解决方案不应被分配到细菌培养, 因为解决方案可能含有漂白剂的痕迹, 可以杀死 OP50。该解决方案也应免除约0.5 厘米远离板块的边缘, 因为鸡蛋可以成为卡在两侧的板块和琼脂。此外, 由于蠕虫的迅速扩散, 将成人转移到新的板块上是将其与后代分离的关键. </li>
	</ol> </li> <li> 分析生存 <ol> <li> 使用生存曲线分析软件分析结果 (参阅材料列表). </li>
		<li> 使用 Grehan-Breslow-魏氏方法比较生存曲线 (假设早期生存时间的数据比以后的时间更准确, 相应地对数据进行加权) 以及 Logrank 统计工具 45 . 
		注意: 例如, 在 图 4B -c 中, 使用变种人 c. 白色念珠菌治疗的蠕虫的生存率与基因野生型控制或 complementarystrains 治疗相比. 
	</ol>
	</li>
</ol>

作者没有什么可透露的。

用于检测线虫感染和生存期的方法对我们所描述的c. 白色念珠菌的影响可以通过修改来测试另一种病原体。另一种细菌或真菌的液体培养物可以用类似的方式制成并喂给C. 线虫。此外, 串行感染可以通过首先暴露幼虫到一个病原体, 然后转移到一个新的板块, 包含一个独立的病原体后, 成年。
在进行这种化验时, 注意时间是很重要的。首先, 当完成鸡蛋准备, ...

线虫已被用作一个强大的模型有机体超过50年。在二十世纪六十年代, 南非生物学家悉尼. 博纳率先使用了线虫来研究神经元的发育, 为长期的科学家们研究线虫的细胞和动物生物学的各个方面铺平了道路。这一血统包括诺贝尔奖得主克雷格. 梅洛和安德鲁火为他们的 rna 干扰工作1, 罗伯特 Horvitz 并且约翰 Sulston 为他们的工作器官发展和细胞凋亡2,3,4, 和马丁查尔菲为他的工作绿色荧光蛋白5。尽管这种模型生物体已经被传统用于研究分子和发育生物学, 但在过去15年中, 研究人员已经开始使用线虫来调查各种人类病原体的生物学, 包括假单胞菌铜绿假单胞菌,金黄色葡萄球菌,沙门氏菌 enterica, 和沙雷菌6,7,89,10。这些研究表明, 许多涉及人与病原体相互作用的机制都是在线虫中保存的, 但也有一些免疫机制是独特的, 这种模式生物体11,12。在自然界中,线虫遇到了来自土壤中的受感染病原体的各种威胁, 这为进化和维持一个复杂的先天免疫系统在其肠道腔内提供了强烈的选择性压力。许多涉及保护肠道腔的基因和机制都是由高度保守的元素组成的, 它们也存在于高哺乳动物中11,13。因此,线虫是研究胃肠病原体的一个很好的模型, 如沙门氏菌 enterica14,痢疾 boydii15, 或霍乱弧菌16。
在这里, 我们强调了显着的多功能性的c. 线虫作为一个模型的宿主, 以研究传染性药物, 如C. 白色念珠菌。C. 线虫作为模型宿主, 可以对毒性进行高通量筛选, 这比小鼠模型更便宜、更耗时, 通常用于研究念珠菌病42。
在这项研究中, 我们表明, 该模型和 assosiated 生存分析可以可靠地用于研究宿主先天免疫效应的重要性, 以抵消感染, 病原体的决定因素驱动毒力, 和药理化合物, 可以干预发病机制。与先前所描述的化验方法不同, 此法提供了一种研究动物生命周期中的病原体, 从幼虫阶段到成年, 而不是仅成年到死亡的手段43,44。总之, 我们的线虫--白色念珠菌模型是一种多功能、功能强大的工具, 不仅可以用于研究驱动感染和免疫的遗传基础, 还可用于识别治疗干预的新化合物。

<table>
	<tbody>
		<tr>
			<td>Agar (granulated, bacterilogical grade)</td>
			<td>Apex BioResearch Products</td>
			<td>20-248</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum Wire (95% Pt, 32 Gauge)</td>
			<td>Genesee Scientific</td>
			<td>59-1M32P</td>
			<td></td>
		</tr>
		<tr>
			<td>Axiovision Zeiss Inverted Microscope</td>
			<td>Axiovision Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto-Peptone</td>
			<td>Fisher BioReagants</td>
			<td>BP1420-500</td>
			<td></td>
		</tr>
		<tr>
			<td>C. elegans strain Bli-3</td>
			<td>Caenorhabditis Genetics Center</td>
			<td>Bli-3(e767) CB767</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Fisher Scientific</td>
			<td>BP51-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholesterol, Sigma Grade, minimum 99%</td>
			<td>Sigma</td>
			<td>C8667-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Culture Tubes (20 x 150 mm)</td>
			<td>FIsherBrand</td>
			<td>14-961-33</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection Microscope (NI-150 High Intensity Illuminator)</td>
			<td>Nikon Instrument Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>E. coli</td>
			<td>Caenorhabditis Genetics Center</td>
			<td>OP50</td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism (Survival Curve Analysis Software)</td>
			<td>GraphPad Software</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LB Broth (Miller&#39;s)</td>
			<td>Apex BioResearch Products</td>
			<td>11-120</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Sulfate</td>
			<td>Fisher Scientific</td>
			<td>10034-99-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Medium Petri Dishes (35 X 10 mm)</td>
			<td>Falcon</td>
			<td>353001</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate monobasic</td>
			<td>Sigma</td>
			<td>P0662-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Scientific</td>
			<td>BP358-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate</td>
			<td>Fisher Scientific</td>
			<td>BP332-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Wildtype C. albicans SC5314</td>
			<td>ATCC</td>
			<td>SC5314</td>
			<td></td>
		</tr>
		<tr>
			<td>Wildtype C. elegans</td>
			<td>Caenorhabditis Genetics Center</td>
			<td>N2</td>
			<td></td>
		</tr>
	</tbody>
</table>

the nematode caenorhabditis elegans - a versatile in vivo model to study host-microbe interactions

我们演示了一种使用线虫线虫作为模型宿主来研究微生物相互作用的方法。通过饮食引入微生物, 使肠道成为疾病的主要部位。线虫肠道结构和功能模仿哺乳动物的肠道, 是透明的, 使其适于对殖民的微观研究。在这里, 我们表明病原体可以导致疾病和死亡。我们能够识别出具有改变毒性的微生物突变体。它对生物胁迫的固有反应使线虫成为一个极好的系统, 可以探测宿主先天免疫相互作用的方方面面。我们发现, 双氧化酶基因突变的寄主不能产生活性氧, 无法抵抗微生物的侮辱。我们进一步证明了所提出的生存试验的通用性, 表明它可以用于研究微生物生长抑制剂的影响。这种方法也可用于发现真菌致病因子作为发展新的抗真菌药物的目标, 以及提供一个机会, 进一步揭示宿主-微生物相互作用。这种检测方法的设计很好地适应了高通量全基因组的屏幕, 而低温保存蠕虫以备将来使用的能力使得它成为一个 cost-effective 和有吸引力的整体动物模型来研究。

病机检测 (图 1) 使用c. 白色念珠菌和c. 线虫以前曾被我们的实验室描述17,18和其他实验室19,,。我们演示了使用可研究 白色念珠菌的毒力, 该病毒显示了c. 白念珠菌的细胞迅速被蠕虫吸收并积聚在肠道内, 导致运动迟缓, 肛门畸形?...

Watch this Scientific Journal Video about The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions at JoVE.com

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

线虫线虫线虫-一个多用途的体内模型研究宿主-微生物相互作用

在这里, 我们提出线虫线虫线虫作为一个多用途的寄主模型来研究微生物的相互作用。

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the ...

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Research

JoVE Journal

Genetics

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

9.3K 次观看.  Worcester Polytechnic Institute. 在这里, 我们提出线虫线虫线虫作为一个多用途的寄主模型来研究微生物的相互作用。

视频: The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

Gene deletion plays an important role in the analysis of gene function. One of the most efficient methods to disrupt genes in a targeted manner is the replacement of the entire gene with a selectable marker via homologous recombination. During homologous recombination, exchange of DNA takes place between sequences with high similarity. Therefore, linear genomic sequences flanking a target gene can be used to specifically direct a selectable marker to the desired integration site. Blunt ends of the deletion construct activate the cell&#39;s DNA repair systems and thereby promote integration of the construct either via homologous recombination or by non-homologous-end-joining. In organisms with efficient homologous recombination, the rate of successful gene deletion can reach more than 50% making this strategy a valuable gene disruption system. The smut fungus Ustilago maydis is a eukaryotic model microorganism showing such efficient homologous recombination. Out of its about 6,900 genes, many have been functionally characterized with the help of deletion mutants, and repeated failure of gene replacement attempts points at essential function of the gene. Subsequent characterization of the gene function by tagging with fluorescent markers or mutations of predicted domains also relies on DNA exchange via homologous recombination. Here, we present the U. maydis strain generation strategy in detail using the simplest example, the gene deletion.

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

We describe a robust gene replacement strategy to genetically manipulate the smut fungus Ustilago maydis. This protocol explains how to generate deletion mutants to investigate infection phenotypes. It can be extended to modify genes in any desired way, e.g., by adding a sequence encoding a fluorescent protein tag.

植物病原体的遗传操作<em&gt;玉米黑粉菌</em&gt;研究真菌生物学和植物微生物相互作用

Gene deletion plays an important role in the analysis of gene function. One of the most efficient methods to disrupt genes in a targeted manner is the ...

科研

遗传学

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 ...

Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia coli, these transfer proteins make up a DNA transfer machinery known as the mating pair formation, or DNA transfer complex, which facilitates conjugative plasmid transfer. The objective of this paper is to provide a method that can be used to determine the role of a specific transfer protein that is involved in conjugation using a series of deletions and/or point mutations in combination with mating assays. The target gene is knocked out on the conjugative plasmid and is then provided in trans through the use of a small recovery plasmid harboring the target gene. Mutations affecting the target gene on the recovery plasmid can reveal information about functional aspects of the target protein that result in the alteration of mating efficiency of donor cells harboring the mutated gene. Alterations in mating efficiency provide insight into the role and importance of the particular transfer protein, or a region therein, in facilitating conjugative DNA transfer. Coupling this mating assay with detailed three-dimensional structural studies will provide a comprehensive understanding of the function of the conjugative transfer protein as well as provide a means for identifying and characterizing regions of protein-protein interaction.

Here, we present a protocol to knockout a gene of interest involved in plasmid conjugation and subsequently analyze the impact of its absence using mating assays. The function of the gene is further explored to a specific region of its sequence using deletion or point mutations.

接合交配测定在接合介入转移蛋白的序列特异性分析

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia ...

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation became a valuable experimental tool to investigate the DNA damage response (DDR) in vivo. It allows real-time high-resolution single-cell analysis of macromolecular dynamics in response to laser-induced damage confined to a submicrometer region in the cell nucleus. However, various laser conditions have been used without appreciation of differences in the types of damage induced. As a result, the nature of the damage is often not well characterized or controlled, causing apparent inconsistencies in the recruitment or modification profiles. We demonstrated that different irradiation conditions (i.e., different wavelengths as well as different input powers (irradiances) of a femtosecond (fs) near-infrared (NIR) laser) induced distinct DDR and repair protein assemblies. This reflects the type of DNA damage produced. This protocol describes how titration of laser input power allows induction of different amounts and complexities of DNA damage, which can easily be monitored by detection of base and crosslinking damages, differential poly (ADP-ribose) (PAR) signaling, and pathway-specific repair factor assemblies at damage sites. Once the damage conditions are determined, it is possible to investigate the effects of different damage complexity and differential damage signaling as well as depletion of upstream factor(s) on any factor of interest.

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

The goal of this protocol is to describe how to use laser microirradiation to induce different types of DNA damage, including relatively simple strand breaks and complex damage, to study DNA damage signaling and repair factor assembly at damage sites in vivo.

激光 Microirradiation 研究体内细胞对简单复杂 DNA 损伤的反应

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation ...

Manipulation of Ploidy in Caenorhabditis elegans

Mechanisms that involve whole genome polyploidy play important roles in development and evolution; also, an abnormal generation of tetraploid cells has been associated with both the progression of cancer and the development of drug resistance. Until now, it has not been feasible to easily manipulate the ploidy of a multicellular animal without generating mostly sterile progeny. Presented here is a simple and rapid protocol for generating tetraploid Caenorhabditis elegans animals from any diploid strain. This method allows the user to create a bias in chromosome segregation during meiosis, ultimately increasing ploidy in C. elegans. This strategy relies on the transient reduction of expression of the rec-8 gene to generate diploid gametes. A rec-8 mutant produces diploid gametes that can potentially produce tetraploids upon fertilization. This tractable scheme has been used to generate tetraploid strains carrying mutations and chromosome rearrangements to gain insight into chromosomal dynamics and interactions during pairing and synapsis in meiosis. This method is efficient for generating stable tetraploid strains without genetic markers, can be applied to any diploid strain, and can be used to derive triploid C. elegans. This straightforward method is useful for investigating other fundamental biological questions relevant to genome instability, gene dosage, biological scaling, extracellular signaling, adaptation to stress, development of resistance to drugs, and mechanisms of speciation.

Manipulation of Ploidy in Caenorhabditis elegans

This method allows for the generation of tetraploid and triploid Caenorhabditis nematodes from any diploid strain. Polyploid strains generated by this method have been used to study chromosome interactions in meiotic prophase, and this method is useful for examining important basic questions in cell, developmental, evolutionary, and cancer biology.

在秀丽线虫中对倍体的操控

Mechanisms that involve whole genome polyploidy play important roles in development and evolution; also, an abnormal generation of tetraploid cells has ...

The Replica Set Method: A High-throughput Approach to Quantitatively Measure Caenorhabditis elegans Lifespan

The Replica Set method is an approach to quantitatively measure lifespan or survival of Caenorhabditis elegans nematodes in a high-throughput manner, thus allowing a single investigator to screen more treatments or conditions over the same amount of time without loss of data quality. The method requires common equipment found in most laboratories working with C. elegans and is thus simple to adopt. The approach centers on assaying independent samples of a population at each observation point, rather than a single sample over time as with traditional longitudinal methods. Scoring entails adding liquid to the wells of a multi-well plate, which stimulates C. elegans to move and facilitates quantifying changes in healthspan. Other major benefits of the Replica Set method include reduced exposure of agar surfaces to airborne contaminants (e.g. mold or fungus), minimal handling of animals, and robustness to sporadic mis-scoring (such as calling an animal as dead when it is still alive). To appropriately analyze and visualize the data from a Replica Set style experiment, a custom software tool was also developed. Current capabilities of the software include plotting of survival curves for both Replica Set and traditional (Kaplan-Meier) experiments, as well as statistical analysis for Replica Set. The protocols provided here describe the traditional experimental approach and the Replica Set method, as well as an overview of the corresponding data analysis.

The Replica Set Method: A High-throughput Approach to Quantitatively Measure Caenorhabditis elegans Lifespan

Here we describe the Replica Set method, an approach to quantitatively measure C. elegans lifespan/survival and healthspan in a high-throughput and robust manner, thus allowing screening of many conditions without sacrificing data quality. This protocol details the strategy and provides a software tool for analysis of Replica Set data.

复制集方法: 一种高通量方法定量测量秀丽线虫寿命

The Replica Set method is an approach to quantitatively measure lifespan or survival of Caenorhabditis elegans nematodes in a high-throughput manner, thus ...

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and effect sizes of regulatory elements to a target gene. Some progress has been made with the bioinformatic prediction of the existence and function of proximal epigenetic modifications associated with activated gene expression using conserved transcription factor binding sites. Chromatin conformation capture studies have revolutionized our ability to discover physical chromatin contacts between sequences and even within an entire genome. Circular chromatin conformation capture coupled with next-generation sequencing (4C-seq), in particular, is designed to discover all possible physical chromatin interactions for a given sequence of interest (viewpoint), such as a target gene or a regulatory enhancer. Current 4C-seq strategies directly sequence from within the viewpoint but require numerous and diverse viewpoints to be simultaneously sequenced to avoid the technical challenges of uniform base calling (imaging) with next generation sequencing platforms. This volume of experiments may not be practical for many laboratories. Here, we report a modified approach to the 4C-seq protocol that incorporates both an additional restriction enzyme digest and qPCR-based amplification steps that are designed to facilitate a greater capture of diverse sequence reads and mitigate the potential for PCR bias, respectively. Our modified 4C method is amenable to the standard molecular biology lab for assessing chromatin architecture.

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

The identification of physical interactions between genes and regulatory elements is challenging but has been facilitated by chromosome conformation capture methods. This modification to the 4C-seq protocol mitigates PCR bias by minimizing over-amplification of PCR templates and maximizes the mappability of reads by incorporating an addition restriction enzyme digest step.

利用下一代序列染色体构象捕获 (4 c 序列) 的高通量识别基因调控数列

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

Advances in sequencing technology have made whole-genome and whole-exome datasets more accessible for both clinical diagnosis and cutting-edge human genetics research. Although a number of in silico algorithms have been developed to predict the pathogenicity of variants identified in these datasets, functional studies are critical to determining how specific genomic variants affect protein function, especially for missense variants. In the Undiagnosed Diseases Network (UDN) and other rare disease research consortia, model organisms (MO) including Drosophila, C. elegans, zebrafish, and mice are actively used to assess the function of putative human disease-causing variants. This protocol describes a method for the functional assessment of rare human variants used in the Model Organisms Screening Center Drosophila Core of the UDN. The workflow begins with gathering human and MO information from multiple public databases, using the MARRVEL web resource to assess whether the variant is likely to contribute to a patient&#39;s condition as well as design effective experiments based on available knowledge and resources. Next, genetic tools (e.g., T2A-GAL4 and UAS-human cDNA lines) are generated to assess the functions of variants of interest in Drosophila. Upon development of these reagents, two-pronged functional assays based on rescue and overexpression experiments can be performed to assess variant function. In the rescue branch, the endogenous fly genes are &#34;humanized&#34;&#160;by replacing the orthologous Drosophila gene with reference or variant human transgenes. In the overexpression branch, the reference and variant human proteins are exogenously driven in a variety of tissues. In both cases, any scorable phenotype (e.g., lethality, eye morphology, electrophysiology) can be used as a read-out, irrespective of the disease of interest. Differences observed between reference and variant alleles suggest a variant-specific effect, and thus likely pathogenicity. This protocol allows rapid, in vivo assessments of putative human disease-causing variants of genes with known and unknown functions.

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

The goal of this protocol is to outline the design and performance of in vivo experiments in Drosophila melanogaster to assess the functional consequences of rare gene variants associated with human diseases.

在Vivo功能研究的疾病相关的稀有人类变异使用果蝇

Advances in sequencing technology have made whole-genome and whole-exome datasets more accessible for both clinical diagnosis and cutting-edge human ...

In Vitro Biochemical Assays using Biotin Labels to Study Protein-Nucleic Acid Interactions

Protein-nucleic acid interactions play important roles in biological processes such as transcription, recombination, and RNA metabolism. Experimental methods to study protein-nucleic acid interactions require the use of fluorescent tags, radioactive isotopes, or other labels to detect and analyze specific target molecules. Biotin, a non-radioactive nucleic acid label, is commonly used in electrophoretic mobility shift assays (EMSA) but has not been regularly employed to monitor protein activity during nucleic acid processes. This protocol illustrates the utility of biotin labeling during in vitro enzymatic reactions, demonstrating that this label works well with a range of different biochemical assays. Specifically, in alignment with previous findings using radioisotope 32P-labeled substrates, it is confirmed via biotin-labeled EMSA that MEIOB (a protein specifically involved in the meiotic recombination) is a DNA-binding protein, that MOV10 (an RNA helicase) resolves biotin-labeled RNA duplex structures, and that MEIOB cleaves biotin-labeled single-stranded DNA. This study demonstrates that biotin is capable of substituting 32P in various nucleic acid-related biochemical assays in vitro.&#160;

Presented here are protocols for in vitro biochemical assays using biotin labels that may be widely&#160;applicable for studying protein-nucleic acid interactions.

使用生物素标签研究蛋白质-核酸相互作用的体外生化分析

Protein-nucleic acid interactions play important roles in biological processes such as transcription, recombination, and RNA metabolism. Experimental ...

Culture and Assay of Large-Scale Mixed-Stage Caenorhabditis elegans Populations

Caenorhabditis elegans (C. elegans) has been and remains a valuable model organism to study developmental biology, aging, neurobiology, and genetics. The large body of work on C. elegans makes it an ideal candidate to integrate into large-population, whole-animal studies to dissect the complex biological components and their relationships with another organism. In order to use C. elegans in collaborative -omics research, a method is needed to generate large populations of animals where a single sample can be split and assayed across diverse platforms for comparative analyses.
Here, a method to culture and collect an abundant mixed-stage C. elegans population on a large-scale culture plate (LSCP) and subsequent phenotypic data is presented. This pipeline yields sufficient numbers of animals to collect phenotypic and population data, along with any data needed for -omics experiments (i.e., genomics, transcriptomics, proteomics, and metabolomics). In addition, the LSCP method requires minimal manipulation to the animals themselves, less user preparation time, provides tight environmental control, and ensures that handling of each sample is consistent throughout the study for overall reproducibility. Lastly, methods to document population size and population distribution of C. elegans life stages in a given LSCP are presented.

Culture and Assay of Large-Scale Mixed-Stage Caenorhabditis elegans Populations

To use Caenorhabditis elegans (C. elegans) in omics research, a method is needed to generate large populations of worms where a single sample can be measured across platforms for comparative analyses. Here, a method to culture C. elegans populations on large-scale culture plates (LSCPs) and to document population growth is presented.

大规模混合阶段 卡诺哈布迪蒂斯电子 人种群的文化和分析

Caenorhabditis elegans (C. elegans) has been and remains a valuable model organism to study developmental biology, aging, neurobiology, and genetics. The ...

线虫线虫线虫-一个多用途的体内模型研究宿主-微生物相互作用

摘要

探索更多视频

此视频中的章节

植物病原体的遗传操作

光遗传学随机突变用组蛋白miniSOG在

接合交配测定在接合介入转移蛋白的序列特异性分析

激光 Microirradiation 研究体内细胞对简单复杂 DNA 损伤的反应

在秀丽线虫中对倍体的操控

复制集方法: 一种高通量方法定量测量秀丽线虫寿命

利用下一代序列染色体构象捕获 (4 c 序列) 的高通量识别基因调控数列

在Vivo功能研究的疾病相关的稀有人类变异使用果蝇

使用生物素标签研究蛋白质-核酸相互作用的体外生化分析

大规模混合阶段卡诺哈布迪蒂斯电子人种群的文化和分析

线虫线虫线虫-一个多用途的体内模型研究宿主-微生物相互作用

摘要

探索更多视频

此视频中的章节

植物病原体的遗传操作

光遗传学随机突变用组蛋白miniSOG在

接合交配测定在接合介入转移蛋白的序列特异性分析

激光 Microirradiation 研究体内细胞对简单复杂 DNA 损伤的反应

在秀丽线虫中对倍体的操控

复制集方法: 一种高通量方法定量测量秀丽线虫寿命

利用下一代序列染色体构象捕获 (4 c 序列) 的高通量识别基因调控数列

在Vivo功能研究的疾病相关的稀有人类变异使用果蝇

使用生物素标签研究蛋白质-核酸相互作用的体外生化分析

大规模混合阶段 卡诺哈布迪蒂斯电子 人种群的文化和分析

大规模混合阶段卡诺哈布迪蒂斯电子人种群的文化和分析