The work is supported by HHMI. We thank our lab members for their help with testing the protocol and revising the manuscript.

1.在LED照明的构建注:需要的LED设备中总结材料的清单。整个LED的设置是小，可以在实验室中的任何位置，但我们建议它被放置在一个黑暗的房间，以控制蠕虫4的曝光。 <ol><li>连接LED与电缆（ 图1A，D）的控制器。 </li><li>该控制器连接到用一根BNC电缆（ 图1A，C，D）的数字函数发生器/放大器。 </li><li>固定LED灯10厘米级以上使用定制支架（ 图1A）。 注:我们做了与金属零件的持有人。 </li><li>覆盖LED照明的设置部分，但要避免热量堆积（ 图1B），这使得蠕虫病。 注:我们使用一个特制的硬质塑料盖用开放的顶部和底部（ 图1A）。盖不需要诱变本身，而是被用来吨Ø限制了蓝光对周围环境的不必要的暴露。我们建议不要覆盖LED的设置完全（ 图1B），以防止过热的蠕虫。 </li><li>佩戴激光防护眼镜。 注意:我们穿的激光防护眼镜，因为光线是非常光明的。太阳镜可以正常工作。 </li><li>切换LED控制器的杠杆"诠释"。为连续光照（ 图1D），并在最大功率（ 图1C）的65％的设置。 </li><li>使用光度计来测量，其中所述蠕虫使用制造商的协议放置台上的蓝色光的强度。该设置使2.0毫瓦/毫米2下连续照明。 </li></ol> 2.蓝光治疗注:使用应变CZ20310 juSi164 [PMEX-5-HIS-72 :: miniSOG-3&#39;UTR（TBB-2）+的Cb-UNC-119（+）] UNC-119（ED3）III 4位的应变才是可用的。在秀丽隐杆线虫遗传中心（CGC，HTTP://www.cgc.cbs.umn.edu/）。 <ol><li>维护标准线虫生长介质（NGM）黑暗中的他，mSOG株E.板大肠杆菌 OP50以下的标准协议10,11。 注:在环境光照下，juSi164株含不显示自发率4以上的突变率。使用带有卤素灯解剖范围一般不引起突变以及常规应变通道。然而，应注意，以避免不必要的曝光， 例如，保持在一个标准暗内孵化的菌株，或覆盖所述菌株与箔。这是建议的情况下，不必要的突变随时间累积收到后冻结应变等分。 </li><li>妊娠挑选年轻的成年人（约12小时后L4）动物蠕虫挑11。 注:年轻的动物显示较少的诱变作用4，而年长的动物可能有更低的育雏大小。 </li><li>切滤波器p纸张，以适应60毫米板用25毫米×25毫米的方形孔和放置到一个未播种板（ 图1E）。 注:正方形冲头可以使用，但冲头的大小不必是精确的。 </li><li>在滤纸上滴100微升的100mM的CuCl 2的均匀地在平板上的方孔中限制蠕虫。 </li><li>挑妊娠年轻成人雌雄同体的期望数目（参见，例如第3步），并转移到的CuCl 2板的中心。 </li><li>佩戴激光防护眼镜。 </li><li>开关ON LED和函数发生器。 </li><li>切换LED控制器的杠杆"分机"。由函数发生器（ 图1D），以控制它。 </li><li>设置在最大功率的65％的控制器和功能发生器作为正弦波，4赫兹（ 图1C）。 </li><li>放置来自步骤2.5的CuCl 2板而不在舞台上的盖的LED（ 图1A &lt;下/ STRONG&gt;）。 </li><li>照亮与蓝色光的动物30分钟。 </li><li>健康的转移寻找蠕虫到种子板为P 0。 注:P 0的所需数量取决于画面的类型。见一个例子第3步。在P 0蠕虫可能表现为光后立即处理不协调，会随着时间恢复。 </li><li>保持在孵化器或在黑暗中使用覆箔蠕虫;并按照标准程序，开始他们的后代当中2,10筛选所需的表型。 </li></ol> 3.正向遗传筛选的例子注意:这与120单倍体基因组克隆画面的一个例子，以检查是否在LED和应变工作图2示出的程序的示意图。 <ol><li> [第一天]在蓝光治疗，挑5看起来健康蠕虫到NGM板OP50为P 0，让他们在一个20℃的孵化器，并让它们产卵1D（&#39;第一天&#39;板）。 </li><li> [第2天]转第0到一个新的种子板块（"第2天"板）。 </li><li> [第三天]选择，并通过焚烧他们杀上的"第2天"平板P 0。 </li><li> [第四天]从&#39;第一天&#39;接盘30妊娠年轻的成年F1和他们转移到个别板块（第30楼1板的&#39;第一天&#39;）。 </li><li>对于"第2天"板[第五天]重复步骤3.4（第30楼1板的"第2天"）。 </li><li> [Day7-9]检查异常形态和/或相对于野生型菌株（ 图3A）F 2蠕虫行为标准立体显微镜下，当他们变得成年人。 注:遗传突变显示约四分之一与F 2之间的同型蠕虫病毒，当它是隐性的高外显率。然而，一些突变体可以生长缓慢和/或显示部分显性。因此，它有可能小于四分之一突变体可以在找到盘子。 </li><li>单独挑可见表型蠕虫和检查表型的遗传下一代。 </li></ol> 4.外的转基因整合的例子<ol><li>注入感兴趣的DNA导入CZ20310 juSi164 [PMEX -5- HIS-72 :: miniSOG-3&#39;UTR（TBB-2）+的Cb-UNC-119（+）] UNC-119（ED3）和制备转基因蠕虫用以下标准注射方案12,13低传输率（10-30％）。 注:另外，建立外阵列可越界进入juSi164株。为了避免juSi164基因分型，juSi164杂合子可以用作在P 0虽然效率可能被降低。对于juSi164的基因分型方法在第5描述。 </li><li>如步骤2中所述[第一天]治疗20〜转基因动物为P 0与蓝色的光。 注:更多的P 0蠕虫取决于TR是必要的<html> ansmission率。转基因可通过表型或共注射标记12,13来识别。 </li><li>挑选10名健康寻找P 0到个别板块，让他们在一个20℃的孵化器，并让它们产卵几天（P 0车牌） </li><li> [第四天]挑出20转f起每个P 0到板个别板块1蠕虫，并让他们产卵。 （20架F 1×10个P 0 =板200°F 1板总）。 </li><li> 【第七天】元函数1板具有转基因&gt; 75％，F 2。 </li><li>挑5的转基因动物从&gt; 75％的传输速率F 1板（F 2板）。 </li><li> [第十天]保持F 2板100％的传输速率作为潜在整合线。 </li><li>异交用标准程序10检查孟德尔遗传规律，并消除潜在的背景突变的潜在整合线。 </li></ol>乐"&gt; 5。基因分型<ol><li>裂解20使用标准协议14异型杂交F 2蠕虫在裂解缓冲液。 </li><li>使用引物对MosSCI插入9，juSi164，（ 表1）用以下的标准协议14的退火温度为58℃进行PCR。 注意:这是不必要的基因型UNC-119（ED3）因为UNC-119（ED3）可以通过不协调行为除去在以下步骤5.4和5.5的抢救转基因（juSi164）后进行检测。 </li><li>运行琼脂糖凝胶电泳15和检查带大小（ 表1）找到WT为juSi164。 </li><li>检查如果F 2后代瘫痪蠕虫由于UNC-119（ED3）的存在。 </li><li>如果步5.4中发现不协调蠕虫，单数nonuncoordinated蠕虫获得下一代所有nonuncoordinated蠕虫。 </li></ol>

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The authors have nothing to disclose.

在这里，我们描述了使用光遗传学诱变的详细流程的His-mSOG在种系表达并提供一个小规模向前遗传画面的一个例子。相比标准化学诱变，此光遗传学诱变方法具有几个优点。首先，它是无毒的，从而保持实验室人员从有毒的化学诱变剂如EMS，ENU路程，用紫外光（TMP / UV）trimethylpsoralen。其次，诱变的实验程序可用于一个小数目为P 0的动物，并且可以在一小时内完成。第三，光遗传学诱变产?...

向前遗传筛选已广泛用来产生遗传突变破坏在模式生物的各种生物过程，如秀丽隐杆线虫（线虫）1。这些突变体的分析导致功能重要基因的发现及其信号通路1,2。传统上，诱变C.线虫使用诱变化学物质，辐射或转座子2来实现的。化学品，如甲磺酸乙酯（EMS）和N-乙基-N- -nitrosourea（ENU）是对人体有毒;伽玛射线或紫外线（UV）辐射诱变需要特殊的设备;和转座子活性菌株，例如突变株3，可在维护期间造成不必要的突变。我们已经开发出一种简单的方法来诱导用基因编码的光敏剂4遗传突变。 活性氧（ROS）可以破坏DNA 5。微型单线态氧发生器（miniSOG）是一个从所述LOV（光，氧气，和电压）工程106氨基酸拟南芥向光2 6的结构域的绿色荧光蛋白。在暴露于蓝光（〜450纳米），miniSOG产生活性氧包括与FL AVIN单核苷酸单线态氧作为辅因子6-8，这是存在于所有细胞中。我们通过标记miniSOG到组蛋白72，C的C末端构成一的His-mSOG融合蛋白组蛋白3. 线虫变种我们产生了一个单拷贝转基因9表达他-mSOG在C的生殖细胞线虫，在黑暗中正常培养条件下，他的-mSOG转基因蠕虫具有正常的育雏大小和寿命4。在暴露于蓝光LED灯，在他的-mSOG蠕虫产生它们的后代中4遗传突变。诱发突变的频谱包括核苷酸变化，如G:C至T的:A和G:C 1至C:G和小chromoso我删除4。这种诱变方法是简单的执行，且只需最少实验室安全设置。这里，我们描述了LED照明设备和程序为光遗传学诱变。

<table border="0" cellpadding="0" cellspacing="0" width="626">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:256px;">Ultra High Power LED light source</td>
			<td style="width:71px;">Prizmatix</td>
			<td style="width:115px;">UHP-mic-LED-460</td>
			<td style="width:184px;">460 &#177; 5 nm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">LED controller</td>
			<td style="width:71px;">Prizmatix</td>
			<td style="width:115px;">UHPLCC-01</td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:256px;">Digital function generator/amplifier</td>
			<td style="width:71px;">PASCO</td>
			<td style="width:115px;">PI-9587C</td>
			<td style="width:184px;">PI-9587C is no longer available. The replacement is PI-8127.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:256px;">BNC cable male/male</td>
			<td style="width:71px;">THORLABS</td>
			<td style="width:115px;">CA3136</td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">USB-TTL interface</td>
			<td style="width:71px;">Prizmatix</td>
			<td style="width:115px;"></td>
			<td style="width:184px;">Optional</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:256px;">Photometer</td>
			<td style="width:71px;">THORLABS</td>
			<td style="width:115px;">PM50 and Model D10MM</td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Filter paper</td>
			<td style="width:71px;">Whatman</td>
			<td style="width:115px;">1001-110</td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:256px;">Copper chloride dihydrate (CuCl2&#8729;2H2O)</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:115px;">C6641</td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Stereomicroscope</td>
			<td style="width:71px;">Leica</td>
			<td style="width:115px;">MZ95</td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="163">
			<td height="163" style="height:163px;width:256px;">NGM plate</td>
			<td style="width:71px;"></td>
			<td style="width:115px;"></td>
			<td style="width:184px;">Dissolve 5g NaCl, 2.5g Peptone, 20g Agar, 10 &#181;g/ml cholesterol in 1 L H2O. After autoclaving, add 1 mM CaCl2, 1 mM MgSO4, 25 mM KH2PO4(pH6.0).&#160;</td>
		</tr>
		<tr height="88">
			<td height="88" style="height:88px;width:256px;">Lysis solution</td>
			<td style="width:71px;"></td>
			<td style="width:115px;"></td>
			<td style="width:184px;">10 mM Tris(pH8.8), 50 mM KCl, 0.1% Triton X100, 2.5 mM MgCl2, 100 &#181;g/ml Proteinase K</td>
		</tr>
	</tbody>
</table>

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.

我们用下面的第2和第3，其中对应于120诱变单倍体基因组的60架F 1板中描述的标准协议，30分钟曝光进行与CZ20310 juSi164 UNC-119（ED3）正向遗传筛选，我们发现8 F 1板带F 2蠕虫显示可见的表型，如身体尺寸缺陷（ 图3B），运动不协调（ 图3C），和成人杀伤力（ 图3D）。我们没有注意到在&#39;第一天"和"第2天"板...

Watch this Scientific Journal Video about Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans at JoVE.com

在秀丽隐杆线虫中使用组蛋白-miniSOG 进行光遗传学随机诱变遗传学 |视频

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

基因编码的蛋白，miniSOG诱导蓝光依赖性全基因组的遗传突变。这种突变方法简单，快速，免费的有毒化学物质，并非常适用于正向遗传学筛查和转基因整合。

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 ...

optogenetic-random-mutagenesis-using-histone-minisog-in-c-elegans

Universidad de Cartagena UDEC

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Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Classical and Modern Genetics

SCIENTISTS IN ACTION

9.0K 次观看.  University of California in San Diego and Howard Hughes Medical Institute. 基因编码的蛋白，miniSOG诱导蓝光依赖性全基因组的遗传突变。这种突变方法简单，快速，免费的有毒化学物质，并非常适用于正向遗传学筛查和转基因整合。 

视频: Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting mutant alleles reside at their endogenous genomic sites and no exogenous DNA sequences are left in the genome following the procedure. Since in haploid yeast cells each of the four core histone proteins is encoded by two non-allelic genes with highly homologous open reading frames (ORFs), targeting mutagenesis specifically to one of two genes encoding a particular histone protein can be problematic. The strategy we describe here bypasses this problem by utilizing sequences outside, rather than within, the ORF of the target genes for the homologous recombination step. Another feature of this system is that the regions of DNA driving the homologous recombination steps can be made to be very extensive, thus increasing the likelihood of successful integration events. These features make this strategy particularly well-suited for histone gene mutagenesis, but can also be adapted for mutagenesis of other genes in the yeast genome.

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

A strategy for generating mutations in histone genes at their endogenous location in Saccharomyces cerevisiae is presented.

目标<em&gt;原位</em&gt;组蛋白突变的基因在芽殖酵母

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting ...

科研

遗传学

Transcriptomic Analysis of C. elegans RNA Sequencing Data Through the Tuxedo Suite on the Galaxy Project

Next generation sequencing (NGS) technologies have revolutionized the nature of biological investigation. Of these, RNA Sequencing (RNA-Seq) has emerged as a powerful tool for gene-expression analysis and transcriptome mapping. However, handling RNA-Seq datasets requires sophisticated computational expertise and poses inherent challenges for biology researchers. This bottleneck has been mitigated by the open access Galaxy project that allows users without bioinformatics skills to analyze RNA-Seq data, and the Database for Annotation, Visualization, and Integrated Discovery (DAVID), a Gene Ontology (GO) term analysis suite that helps derive biological meaning from large data sets. However, for first-time users and bioinformatics' amateurs, self-learning and familiarization with these platforms can be time-consuming and daunting. We describe a straightforward workflow that will help C. elegans researchers to isolate worm RNA, conduct an RNA-Seq experiment and analyze the data using Galaxy and DAVID platforms. This protocol provides stepwise instructions for using the various Galaxy modules for accessing raw NGS data, quality-control checks, alignment, and differential gene expression analysis, guiding the user with parameters at every step to generate a gene list that can be screened for enrichment of gene classes or biological processes using DAVID. Overall, we anticipate that this article will provide information to C. elegans researchers undertaking RNA-Seq experiments for the first time as well as frequent users running a small number of samples.

Transcriptomic Analysis of C. elegans RNA Sequencing Data Through the Tuxedo Suite on the Galaxy Project

Galaxy and DAVID have emerged as popular tools that allow investigators without bioinformatics training to analyze and interpret RNA-Seq data. We describe a protocol for C. elegans researchers to perform RNA-Seq experiments, access and process the dataset using Galaxy and obtain meaningful biological information from the gene lists using DAVID.

的转录分析<em&gt;ç</em&gt;。<em&gt;线虫</em&gt; RNA测序数据通过炫套房的银河项目

Next generation sequencing (NGS) technologies have revolutionized the nature of biological investigation. Of these, RNA Sequencing (RNA-Seq) has emerged ...

Analysis of the c-KIT Ligand Promoter Using Chromatin Immunoprecipitation

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. Therefore, DNA-protein interactions regulate multiple physiological, pathophysiological, and biological functions, such as cell differentiation, cell proliferation, cell cycle control, chromosome stability, epigenetic gene regulation, and cell transformation. In eukaryotic cells, the DNA interacts with histone and nonhistone proteins and is condensed into chromatin. Several technical tools can be used to analyze DNA-protein interactions, such as the Electrophoresis (gel) Mobility Shift Assay (EMSA) and DNase I footprinting. However, these techniques analyze the protein-DNA interaction in vitro, not within the cellular context. Chromatin immunoprecipitation (ChIP) is a technique that captures proteins at their specific DNA binding sites, thereby allowing for the identification of DNA-protein interactions within their chromatin context. It is done by fixation&#160;of the DNA-protein interaction, followed by&#160;immunoprecipitation of the protein of interest. Subsequently, the genomic site that the protein was bound to is characterized. Here, we describe and discuss ChIP and demonstrate its analytical value for the identification of the Transforming Growth Factor-&#946; (TGF-&#946;)-induced binding of the transcription factor SMAD2 to SMAD Binding Elements (SBE) within the promoter region of the tyrosine-protein kinase Kit (c-KIT) receptor ligand Stem Cell Factor (SCF).

DNA-protein interactions are essential for multiple biological processes. During the evaluation of cellular functions, the analysis of DNA-protein interactions is indispensable for understanding gene regulation. Chromatin immunoprecipitation (ChIP) is a powerful tool to analyze such interactions in vivo.

使用染色质免疫沉淀分析c-KIT配体启动子

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. ...

Protocols for C-Brick DNA Standard Assembly Using Cpf1

CRISPR-associated protein Cpf1 cleaves double-stranded DNA under the guidance of CRISPR RNA (crRNA), generating sticky ends. Because of this characteristic, Cpf1 has been used for the establishment of a DNA assembly standard called C-Brick, which has the advantage of long recognition sites and short scars. On a standard C-Brick vector, there are four Cpf1 recognition sites &#8211; the prefix (T1 and T2 sites) and the suffix (T3 and T4 sites) &#8211; flanking biological DNA parts. The cleavage of T2 and T3 sites produces complementary sticky ends, which allow for the assembly of DNA parts with T2 and T3 sites. Meanwhile, a short &#34;GGATCC&#34; scar is generated between parts after assembly. As the newly formed plasmid once again contains the four Cpf1 cleavage sites, the method allows for the iterative assembly of DNA parts, which is similar to those of BioBrick and BglBrick standards. A procedure outlining the use of the C-Brick standard to assemble DNA parts is described here. The C-Brick standard can be widely used by scientists, graduate and undergraduate students, and even amateurs.

CRISPR-associated protein Cpf1 can be guided by a specially designed CRISPR RNA (crRNA) to cleave double-stranded DNA at desired sites, generating sticky ends. Based on this characteristic, a DNA assembly standard (C-Brick) was established, and a protocol detailing its use is described here.

使用Cpf1的C-Brick DNA标准装配的方案

CRISPR-associated protein Cpf1 cleaves double-stranded DNA under the guidance of CRISPR RNA (crRNA), generating sticky ends. Because of this ...

Quantification of Information Encoded by Gene Expression Levels During Lifespan Modulation Under Broad-range Dietary Restriction in C. elegans

Sensory systems allow animals to detect, process, and respond to their environment. Food abundance is an environmental cue that has profound effects on animal physiology and behavior. Recently, we showed that modulation of longevity in the nematode Caenorhabditis elegans by food abundance is more complex than previously recognized. The responsiveness of the lifespan to changes in food level is determined by specific genes that act by controlling information processing within a neural circuit. Our framework combines genetic analysis, high-throughput quantitative imaging and information theory. Here, we describe how these techniques can be used to characterize any gene that has a physiological relevance to broad-range dietary restriction. Specifically, this workflow is designed to reveal how a gene of interest regulates lifespan under broad-range dietary restriction; then to establish how the expression of the gene varies with food level; and finally, to provide an unbiased quantification of the amount of information conveyed by gene expression about food abundance in the environment. When several genes are examined simultaneously under the context of a neural circuit, this workflow can uncover the coding strategy employed by the circuit.

Quantification of Information Encoded by Gene Expression Levels During Lifespan Modulation Under Broad-range Dietary Restriction in C. elegans

Here, we present a framework to relate broad-range dietary restriction to gene expression and lifespan. We describe protocols for broad-range dietary restriction and for quantitative imaging of gene expression under this paradigm. We further outline computational analyses to reveal underlying information processing features of the genetic circuits involved in food-sensing.

量化的信息编码的基因表达水平在寿命调制下线虫的宽范围饮食限制

Sensory systems allow animals to detect, process, and respond to their environment. Food abundance is an environmental cue that has profound effects on ...

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 ...

Gene-targeted Random Mutagenesis to Select Heterochromatin-destabilizing Proteasome Mutants in Fission Yeast

Random mutagenesis of a target gene is commonly used to identify mutations that yield the desired phenotype. Of the methods that may be used to achieve random mutagenesis, error-prone PCR is a convenient and efficient strategy for generating a diverse pool of mutants (i.e., a mutant library). Error-prone PCR is the method of choice when a researcher seeks to mutate a pre-defined region, such as the coding region of a gene while leaving other genomic regions unaffected. After the mutant library is amplified by error-prone PCR, it must be cloned into a suitable plasmid. The size of the library generated by error-prone PCR is constrained by the efficiency of the cloning step. However, in the fission yeast, Schizosaccharomyces pombe, the cloning step can be replaced by the use of a highly efficient one-step fusion PCR to generate constructs for transformation. Mutants of desired phenotypes may then be selected using appropriate reporters. Here, we describe this strategy in detail, taking as an example, a reporter inserted at centromeric heterochromatin.

This article describes a detailed methodology for random mutagenesis of a target gene in fission yeast. As an example, we target rpt4+, which encodes a subunit of the 19S proteasome, and screen for mutations that destabilize heterochromatin.

基因靶向随机诱变在裂变酵母中选择染色质稳定蛋白酶突变体

Random mutagenesis of a target gene is commonly used to identify mutations that yield the desired phenotype. Of the methods that may be used to achieve ...

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

The clustered regularly interspersed palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) prokaryotic adaptive immune defense system has been co-opted as a powerful tool for precise eukaryotic genome engineering. Here, we present a rapid and simple method using chimeric single guide RNAs (sgRNA) and CRISPR-Cas9 Ribonucleoproteins (RNPs) for the efficient and precise generation of genomic point mutations in C. elegans. We describe a pipeline for sgRNA target selection, homology-directed repair (HDR) template design, CRISPR-Cas9-RNP complexing and delivery, and a genotyping strategy that enables the robust and rapid identification of correctly edited animals. Our approach not only permits the facile generation and identification of desired genomic point mutant animals, but also facilitates the detection of other complex indel alleles in approximately 4 - 5 days with high efficiency and a reduced screening workload.

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

Here, we present a method to engineer the genome of C. elegans using CRISPR-Cas9 ribonucleoproteins and homology dependent repair templates.

利用 CRISPR/Cas9 Ribonucleoproteins 快速简便地生成线虫基因组点突变体的管道

The clustered regularly interspersed palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) prokaryotic adaptive immune defense system has been ...

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

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

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

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

用细胞遗传学技术测定芽孢杆菌的受精率

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

High-Throughput Quantitative RT-PCR in Single and Bulk C. elegans Samples Using Nanofluidic Technology

This paper presents a high-throughput reverse transcription quantitative PCR (RT-qPCR) assay for Caenorhabditis elegans that is fast, robust, and highly sensitive. This protocol obtains precise measurements of gene expression from single worms or from bulk samples. The protocol presented here provides a novel adaptation of existing methods for complementary DNA (cDNA) preparation coupled to a nanofluidic RT-qPCR platform. The first part of this protocol, named &#8216;Worm-to-CT&#8217;, allows cDNA production directly from nematodes without the need for prior mRNA isolation. It increases experimental throughput by allowing the preparation of cDNA from 96 worms in 3.5 h. The second part of the protocol uses existing nanofluidic technology to run high-throughput RT-qPCR on the cDNA. This paper evaluates two different nanofluidic chips: the first runs 96 samples and 96 targets, resulting in 9,216 reactions in approximately 1.5 days of benchwork. The second chip type consists of six 12 x 12 arrays, resulting in 864 reactions. Here, the Worm-to-CT method is demonstrated by quantifying mRNA levels of genes encoding heat shock proteins from single worms and from bulk samples. Provided is an extensive list of primers designed to amplify processed RNA for the majority of coding genes within the C. elegans genome.

High-Throughput Quantitative RT-PCR in Single and Bulk C. elegans Samples Using Nanofluidic Technology

In this article a high-throughput protocol for fast and reliable determination of gene expression levels in single or bulk C. elegans samples is described. This protocol does not require RNA isolation and produces cDNA directly from samples. It can be used together with high-throughput multiplexed nanofluidic real-time qPCR platforms.

使用纳米流体技术的单体和散装 C. 电子样品 中的高通量定量 RT-PCR

This paper presents a high-throughput reverse transcription quantitative PCR (RT-qPCR) assay for Caenorhabditis elegans that is fast, robust, and highly ...

光遗传学随机突变用组蛋白miniSOG在

摘要

探索更多视频

此视频中的章节

目标

的转录分析

使用染色质免疫沉淀分析c-KIT配体启动子

使用Cpf1的C-Brick DNA标准装配的方案

量化的信息编码的基因表达水平在寿命调制下线虫的宽范围饮食限制

在秀丽线虫中对倍体的操控

基因靶向随机诱变在裂变酵母中选择染色质稳定蛋白酶突变体

利用 CRISPR/Cas9 Ribonucleoproteins 快速简便地生成线虫基因组点突变体的管道

用细胞遗传学技术测定芽孢杆菌的受精率

使用纳米流体技术的单体和散装 C. 电子样品中的高通量定量 RT-PCR

光遗传学随机突变用组蛋白miniSOG在

摘要

探索更多视频

此视频中的章节

目标

的转录分析

使用染色质免疫沉淀分析c-KIT配体启动子

使用Cpf1的C-Brick DNA标准装配的方案

量化的信息编码的基因表达水平在寿命调制下线虫的宽范围饮食限制

在秀丽线虫中对倍体的操控

基因靶向随机诱变在裂变酵母中选择染色质稳定蛋白酶突变体

利用 CRISPR/Cas9 Ribonucleoproteins 快速简便地生成线虫基因组点突变体的管道

用细胞遗传学技术测定芽孢杆菌的受精率

使用纳米流体技术的单体和散装 C. 电子样品 中的高通量定量 RT-PCR

使用纳米流体技术的单体和散装 C. 电子样品中的高通量定量 RT-PCR