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>

<ol><li>Jorgensen, E. M., Mango, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+art+and+design+of+genetic+screens%3A+caenorhabditis+elegans%5BTitle%5D)+AND+%22Nat+Rev+Genet%22%5BJournal%5D)">The art and design of genetic screens: caenorhabditis elegans.</a> Nat Rev Genet. 3 (5), 356-369 (2002).</li>
<li>Kutscher, L. M., Shaham, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Forward+and+reverse+mutagenesis+in+C.+elegans%5BTitle%5D)+AND+%22WormBook%22%5BJournal%5D)">Forward and reverse mutagenesis in C. elegans.</a> WormBook. , 1-26 (2014).</li>
<li>Collins, J., Saari, B., Anderson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Activation+of+a+transposable+element+in+the+germ+line+but+not+the+soma+of+Caenorhabditis+elegans%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Activation of a transposable element in the germ line but not the soma of Caenorhabditis elegans.</a> Nature. 328 (6132), 726-728 (1987).</li>
<li>Noma, K., Jin, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optogenetic+mutagenesis+in+Caenorhabditis+elegans%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Optogenetic mutagenesis in Caenorhabditis elegans.</a> Nat Commun. 6, 8868(2015).</li>
<li>Cooke, M. S., Evans, M. D., Dizdaroglu, M., Lunec, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Oxidative+DNA+damage%3A+mechanisms%2C+mutation%2C+and+disease%5BTitle%5D)+AND+%22FASEB+J%22%5BJournal%5D)">Oxidative DNA damage: mechanisms, mutation, and disease.</a> FASEB J. 17 (10), 1195-1214 (2003).</li>
<li>Shu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+genetically+encoded+tag+for+correlated+light+and+electron+microscopy+of+intact+cells%2C+tissues%2C+and+organisms%5BTitle%5D)+AND+%22PLoS+Biol%22%5BJournal%5D)">A genetically encoded tag for correlated light and electron microscopy of intact cells, tissues, and organisms.</a> PLoS Biol. 9 (4), 1001041(2011).</li>
<li>Ruiz-Gonzalez, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Singlet+oxygen+generation+by+the+genetically+encoded+tag+miniSOG%5BTitle%5D)+AND+%22J+Am+Chem+Soc%22%5BJournal%5D)">Singlet oxygen generation by the genetically encoded tag miniSOG.</a> J Am Chem Soc. 135 (26), 9564-9567 (2013).</li>
<li>Pimenta, F. M., Jensen, R. L., Breitenbach, T., Etzerodt, M., Ogilby, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Oxygen-dependent+photochemistry+and+photophysics+of+%22miniSOG%2C%22+a+protein-encased+flavin%5BTitle%5D)+AND+%22Photochem+Photobiol%22%5BJournal%5D)">Oxygen-dependent photochemistry and photophysics of "miniSOG," a protein-encased flavin.</a> Photochem Photobiol. 89 (5), 1116-1126 (2013).</li>
<li>Frokjaer-Jensen, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Random+and+targeted+transgene+insertion+in+Caenorhabditis+elegans+using+a+modified+Mos1+transposon%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">Random and targeted transgene insertion in Caenorhabditis elegans using a modified Mos1 transposon.</a> Nat Methods. 11 (5), 529-534 (2014).</li>
<li>Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+genetics+of+Caenorhabditis+elegans%5BTitle%5D)+AND+%22Genetics%22%5BJournal%5D)">The genetics of Caenorhabditis elegans.</a> Genetics. 77 (1), 71-94 (1974).</li>
<li>Chaudhuri, J., Parihar, M., Pires-daSilva, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+introduction+to+worm+lab%3A+from+culturing+worms+to+mutagenesis%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">An introduction to worm lab: from culturing worms to mutagenesis.</a> J Vis Exp. (47), (2011).</li>
<li>Berkowitz, L. A., Knight, A. L., Caldwell, G. A., Caldwell, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generation+of+stable+transgenic+C.+elegans+using+microinjection%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Generation of stable transgenic C. elegans using microinjection.</a> J Vis Exp. (18), (2008).</li>
<li>Mello, C., Fire, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((DNA+transformation%5BTitle%5D)+AND+%22Methods+Cell+Biol%22%5BJournal%5D)">DNA transformation.</a> Methods Cell Biol. 48, 451-482 (1995).</li>
<li>Fay, D., Bender, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetic+mapping+and+manipulation%3A+chapter+4--SNPs%3A+introduction+and+two-point+mapping%5BTitle%5D)+AND+%22WormBook%22%5BJournal%5D)">Genetic mapping and manipulation: chapter 4--SNPs: introduction and two-point mapping.</a> WormBook. , 1-7 (2006).</li>
<li>Lee, P. Y., Costumbrado, J., Hsu, C. Y., Kim, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Agarose+gel+electrophoresis+for+the+separation+of+DNA+fragments%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Agarose gel electrophoresis for the separation of DNA fragments.</a> J Vis Exp. (62), (2012).</li>
<li>Westberg, M., Holmegaard, L., Pimenta, F. M., Etzerodt, M., Ogilby, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rational+design+of+an+efficient%2C+genetically+encodable%2C+protein-encased+singlet+oxygen+photosensitizer%5BTitle%5D)+AND+%22J+Am+Chem+Soc%22%5BJournal%5D)">Rational design of an efficient, genetically encodable, protein-encased singlet oxygen photosensitizer.</a> J Am Chem Soc. 137 (4), 1632-1642 (2015).</li>
<li>Xu, S., Chisholm, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Highly+efficient+optogenetic+cell+ablation+in+C.+elegans+using+membrane-targeted+miniSOG%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Highly efficient optogenetic cell ablation in C. elegans using membrane-targeted miniSOG.</a> Sci Rep. 6, 21271(2016).</li>
<li>Mariol, M. C., Walter, L., Bellemin, S., Gieseler, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+rapid+protocol+for+integrating+extrachromosomal+arrays+with+high+transmission+rate+into+the+C.+elegans+genome%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">A rapid protocol for integrating extrachromosomal arrays with high transmission rate into the C. elegans genome.</a> J Vis Exp. (82), (2013).</li>
<li>Lin, J. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optogenetic+inhibition+of+synaptic+release+with+chromophore-assisted+light+inactivation+%28CALI%29%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Optogenetic inhibition of synaptic release with chromophore-assisted light inactivation (CALI).</a> Neuron. 79 (2), 241-253 (2013).</li>
<li>Minevich, G., Park, D. S., Blankenberg, D., Poole, R. J., Hobert, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CloudMap%3A+A+Cloud-Based+Pipeline+for+Analysis+of+Mutant+Genome+Sequences%5BTitle%5D)+AND+%22Genetics%22%5BJournal%5D)">CloudMap: A Cloud-Based Pipeline for Analysis of Mutant Genome Sequences.</a> Genetics. 192 (>4), 1249-1269 (2012).</li>
<li>Qi, Y., Xu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MiniSOG-mediated+Photoablation+in+Caenorhabdtis+elegans%5BTitle%5D)+AND+%22Bio-protocol%22%5BJournal%5D)">MiniSOG-mediated Photoablation in Caenorhabdtis elegans.</a> Bio-protocol. 3 (1), http://www.bio-protocol.org/e316 316(2013).</li>
<li>Qi, Y. B., Garren, E. J., Shu, X., Tsien, R. Y., Jin, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Photo-inducible+cell+ablation+in+Caenorhabditis+elegans+using+the+genetically+encoded+singlet+oxygen+generating+protein+miniSOG%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Photo-inducible cell ablation in Caenorhabditis elegans using the genetically encoded singlet oxygen generating protein miniSOG.</a> Proc Natl Acad Sci U S A. 109 (19), 7499-7504 (2012).</li>
<li>Nagel, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Light+activation+of+channelrhodopsin-2+in+excitable+cells+of+Caenorhabditis+elegans+triggers+rapid+behavioral+responses%5BTitle%5D)+AND+%22Curr+Biol%22%5BJournal%5D)">Light activation of channelrhodopsin-2 in excitable cells of Caenorhabditis elegans triggers rapid behavioral responses.</a> Curr Biol. 15 (24), 2279-2284 (2005).</li>
</ol>

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，光遗传学诱变产生的突变的广谱包括核苷酸取代和染色体缺失。最后，光遗传学诱变可用于外转基因的整合。 在此诱变协议的关键步骤是，以确保LED的设置适用于他的-mSOG含有菌株在离存在意外效率。我们建议进行试点克隆屏幕，在协议第3节所述，大约100单倍体基因组。该突变是通过增加P 0的个数为光治疗从小规模试点屏幕，屏幕nonclonal易于扩展。对于大型nonclonal屏幕，我们建议同步蠕虫妊娠年轻人和转移数百人与M9的解决方案到氯化铜板光治疗。在标准状态中的步骤2和3所述，估计具有比目前广泛使用的50毫EMS诱变法4效率低大约4.4倍。突变率可以通过增加光照射时间和/或增加的光的强度得到改善。但是，这些修改可能会导致光毒性，导致光处理的蠕虫小尺寸育雏和疾病。为了增加效率，它可能会使用miniSOG衍生物具有更高的活性氧的产量，例如作为miniSOG（Q103L），虽然它可能会增加，而不光治疗16,17背景诱变作用。对于该DNA构建体的变型中，PMEX-5-HIS-72 :: miniSOG-3&#39;UTR（TBB-2）的质粒（pCZ886）是可商购。 30％ -下这里所描述的条件下，以平均4％200°F 1大约一行使用具有10的传输速率外的转基因品系，当产生的组合转基因。这种效率可通过使用外转基因品系具有如前对紫外线辐射方法18报高传输速率增加。 他-mSOG介导光遗传学诱变诱导范围广泛的基因突变。此外，位点的使用光遗传学诱变的rpm-1抑制屏幕回收的频率是相似的，从使用EMS 4相同的屏幕。然而，这是possi竹叶提取使用的是组蛋白基因可能对某些基因的偏见，那光遗传学突变，由于未知因素，如染色质结构。为了解决这个问题，有必要分析由全基因组测序许多optogenetically诱导的突变。 他-mSOG介导光遗传学诱变导致突变的广谱包括单核苷酸取代和缺失范围从1bp至几百bp的4。单核苷酸变体的光谱表明miniSOG诱导活性氧是突变4的原因。它也可能是miniSOG损害组蛋白并导致染色质的不稳定，因为miniSOG被用于灭活蛋白质，以及19。有可能通过使用不同强度的光来操作的突变类型。因为光遗传学诱变导致缺失，它分析缺失以及谁单核苷酸变化是非常重要的映射20勒基因组测序数据。 据报道，此前啶显微镜用于miniSOG介导的细胞消融21,22。一个标准啶化合物显微镜的光强度测定为〜0.5毫瓦/毫米2无透镜，这是约4倍比在标准光遗传学诱变协议使用更低。因此，这是非常可取用外延荧光显微镜作为光源时，根据经验确定的效率。在LED的一个优点是在数千小时的稳定性。以前的研究已经表明，miniSOG是在根据脉冲光22 ROS介导的细胞消融更有效，虽然确切的机制仍有待确定。我们使用的数字函数发生器/放大器，使脉冲光。另一种选择是使用USB-TTL接口（ 图1A），以调节LED立光源连接到计算机通过程序GHT。这里所描述的LED系统，也可用于其他目的，例如使用channelrhodopsin 23可兴奋细胞的光遗传学控制，细胞消融使用mitochondria-或细胞膜定位miniSOG 17,22，和生色辅助光灭活（卡利）的蛋白19。此外，该光遗传学诱变已被应用到其他生物，如细菌，酵母，飞行，斑马鱼，以及哺乳动物细胞系的潜力。

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><tbody><tr><td>Ultra High Power LED light source</td><td>Prizmatix</td><td>UHP-mic-LED-460</td><td>460 &amp;plusmn; 5 nm</td></tr><tr><td>LED controller</td><td>Prizmatix</td><td>UHPLCC-01</td><td></td></tr><tr><td>Digital function generator/amplifier</td><td>PASCO</td><td>PI-9587C</td><td>PI-9587C is no longer available. The replacement is PI-8127.</td></tr><tr><td>BNC cable male/male</td><td>THORLABS</td><td>CA3136</td><td></td></tr><tr><td>USB-TTL interface</td><td>Prizmatix</td><td></td><td>Optional</td></tr><tr><td>Photometer</td><td>THORLABS</td><td>PM50 and Model D10MM</td><td></td></tr><tr><td>Filter paper</td><td>Whatman</td><td>1001-110</td><td></td></tr><tr><td>Stereomicroscope</td><td>Leica</td><td>MZ95</td><td></td></tr><tr><td>NGM plate</td><td></td><td></td><td>Dissolve 5 g NaCl, 2.5 g Peptone, 20 g Agar, 10 &amp;micro;g/mL cholesterol in 1 L H&lt;sub&gt;2&lt;/sub&gt;O. After autoclaving, add 1 mM CaCl&lt;sub&gt;2&lt;/sub&gt;, 1 mM MgSO&lt;sub&gt;4&lt;/sub&gt;, 25 mM KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;(pH 6.0).&amp;nbsp;</td></tr><tr><td>Lysis solution</td><td></td><td></td><td>10 mM Tris(pH 8.8), 50 mM KCl, 0.1% Triton X-100, 2.5 mM MgCl&lt;sub&gt;2&lt;/sub&gt;, 100 &amp;micro;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"板之间的突变体的数量的任何差异。单挑小F 2蠕虫和不协调F 2蠕虫所有后代显示出相应的表型，这表明它们是可遗传的突变体。全观察总结于表2，平均为120单倍体基因组的克隆屏幕将导致大约10架F 1板CON泰宁蠕虫具有明显的表型检测，如体形和运动，很容易在立体显微镜下发现。更定量分析是必要的，以确定突变率精确4。可见突变体从该屏幕的预期数量表明，应变和设置是为光遗传学突变正常工作。 <img alt="图1" src="/files/ftp_upload/54810/54810fig1.jpg" /> 图1:LED的设置 （A）整个系统。该LED安装在一个特制的支架。详情在材料列表中找到。 （B）的盖是由塑料制成。它不完全覆盖系统，以避免热积累。 （C）控制器和功能发生器。 （D）的系统的背面。 （ 五 ）非种子板与蓝光治疗氯化铜-soaked滤纸。<html> <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54810/54810fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54810/54810fig2.jpg" /> 图2:用于克隆屏幕实验程序后的蓝色光治疗，P 0蠕虫转移到种子板并使其产卵。 P 0蠕虫光处理后转移到一个新的种子板1 D和光处理后杀死2天。 ˚F1蠕虫从P 0板单挑。 F 2蠕虫表型检查后，他们成为成年人。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54810/54810fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 10fig3.jpg"/&gt; 图3:突变体的示例 （A）为光遗传学突变原始菌株:CZ20310 juSi164 UNC-119（ED3）。该菌株具有正常的身体形态，行为和育雏大小。比例尺:0.5毫米。 。（B - D）F 2后代显示身体尺寸的缺陷（B），不协调（C），和致命的（D）表型<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54810/54810fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="表格1" src="/files/ftp_upload/54810/54810table1.jpg" /> 表1:juSi164和 UNC-119（ED3） 基因分型 。 <img alt="表2" src="/files/ftp_upload/54810/54810table2.jpg" /> 表2:表型fr的实施例OM一个克隆屏幕。

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

Nanyang Technological University

Research

Education

JoVE Journal

JoVE Core

Biology

Genetics

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

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 ...

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

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain and protect cellular identities. The conversion of germ cells into specific neurons in the nematode Caenorhabditis elegans (C. elegans) is a powerful tool for performing genetic screens in order to dissect regulatory pathways that safeguard established cell identities. Reprogramming of germ cells to a specific type of neurons termed ASE requires transgenic animals that allow broad over-expression of the Zn-finger transcription factor (TF) CHE-1. Endogenous CHE-1 is expressed exclusively in two head neurons and is required to specify the glutamatergic ASE neurons fate, which can easily be visualized by the gcy-5prom::gfp reporter. A trans gene containing the heat-shock promoter-driven che-1 gene expression construct allows broad mis-expression of CHE-1 in the entire animal upon heat-shock treatment. The combination of RNAi against the chromatin-regulating factor LIN-53 and heat-shock-induced che-1 over-expression leads to reprogramming of germ cell into ASE neuron-like cells. We describe here the specific RNAi procedure and appropriate conditions for heat-shock treatment of transgenic animals in order to successfully induce germ cell to neuron conversion.

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

This protocol describes how to study cellular processes during cell fate conversion in Caenorhabditis elegans in vivo. Using transgenic animals, allowing heat-shock promoter-driven overexpression of the neuron fate-inducing transcription factor CHE-1 and RNAi-mediated depletion of the chromatin-regulating factor LIN-53 germ cell to neuron reprogramming can be observed in vivo.

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

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

发育生物学

In vivo Neuronal Calcium Imaging in C. elegans

The nematode worm C. elegans is an ideal model organism for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon 1 and GCaMP 2 allow in vivo imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans presents a robust and flexible system for in vivo neuronal imaging with advantages over other model systems in technical simplicity and cost.

In vivo Neuronal Calcium Imaging in C. elegans

With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.

C.在体内神经元钙成像技术线虫

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

神经科学

An Introduction to Worm Lab: from Culturing Worms to Mutagenesis

This protocol describes procedures to maintain nematodes in the laboratory and how to mutagenize them using two alternative methods: ethyl methane sulfonate (EMS) and 4, 5', 8-trimethylpsoralen combined with ultraviolet light (TMP/UV). Nematodes are powerful biological systems for genetics studies because of their simple body plan and mating system, which is composed of self-fertilizing hermaphrodites and males that can generate hundreds of progeny per animal. Nematodes are maintained in agar plates containing a lawn of bacteria and can be easily transferred from one plate to another using a pick. EMS is an alkylating agent commonly used to induce point mutations and small deletions, while TMP/UV mainly induces deletions. Depending on the species of nematode being used, concentrations of EMS and TMP will have to be optimized. To isolate recessive mutations of the nematode Pristionchus pacificus, animals of the F2 generation were visually screened for phenotypes. To illustrate these methods, we mutagenized worms and looked for Uncoordinated (Unc), Dumpy (Dpy) and Transformer (Tra) mutants.

Screening for mutants with phenotypic defects is a straightforward method for identifying genes that function in a given biological process. In this article we describe how to culture free living worms (e.g., Pristionchus pacificus) in the laboratory and show two different mutagenesis methods, EMS and TMP/UV.

蠕虫病毒实验室介绍：从培养蠕虫突变

This protocol describes procedures to maintain nematodes in the laboratory and how to mutagenize them using two alternative methods: ethyl methane ...

生物学

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using fluorescent-labeled reporter proteins or differential interference contrast (DIC) microscopy, allows for the examination of the temporal progression of these dynamic events which is otherwise inferred from analysis of fixed samples1,2. Moreover, the study of the developmental regulations of cellular processes necessitates conducting time-lapse experiments on an intact organism during development. The Caenorhabiditis elegans embryo is light-transparent and has a rapid, invariant developmental program with a known cell lineage3, thus providing an ideal experiment model for studying questions in cell biology4,5and development6-9. C. elegans is amendable to genetic manipulation by forward genetics (based on random mutagenesis10,11) and reverse genetics to target specific genes (based on RNAi-mediated interference and targeted mutagenesis12-15). In addition, transgenic animals can be readily created to express fluorescently tagged proteins or reporters16,17. These traits combine to make it easy to identify the genetic pathways regulating fundamental cellular and developmental processes in vivo18-21. In this protocol we present methods for live imaging of C. elegans embryos using DIC optics or GFP fluorescence on a compound epifluorescent microscope. We demonstrate the ease with which readily available microscopes, typically used for fixed sample imaging, can also be applied for time-lapse analysis using open-source software to automate the imaging process.

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

The C. elegans embryo is a powerful system for studying cell biology and development. We present a protocol for live imaging of C. elegans embryos utilizing DIC optics or fluorescence using readily available epifluorescent microscopes and open-source software.

成像 C。线虫胚胎使用啶显微镜和开放源码软件

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using ...

A Rapid Protocol for Integrating Extrachromosomal Arrays With High Transmission Rate into the C. elegans Genome

Microinjecting DNA into the cytoplasm of the syncytial gonad of Caenorhabditis elegans is the main technique used to establish transgenic lines that exhibit partial and variable transmission rates of extrachromosomal arrays to the next generation. In addition, transgenic animals are mosaic and express the transgene in a variable number of cells. Extrachromosomal arrays can be integrated into the C. elegans genome using UV irradiation to establish nonmosaic transgenic strains with 100% transmission rate of the transgene. To that extent, F1 progenies of UV irradiated transgenic animals are screened for animals carrying a heterozygous integration of the transgene, which leads to a 75% Mendelian transmission rate to the F2 progeny. One of the challenges of this method is to distinguish between the percentage of transgene transmission in a population before (X% transgenic animals) and after integration (&#8805;75% transgenic F2 animals). Thus, this method requires choosing a nonintegrated transgenic line with a percentage of transgenic animals that is significantly lower than the Mendelian segregation of 75%. Consequently, nonintegrated transgenic lines with an extrachromosomal array transmission rate to the next generation &#8804;60% are usually preferred for integration, and transgene integration in highly transmitting strains is difficult. Here we show that the efficiency of extrachromosomal arrays integration into the genome is increased when using highly transmitting transgenic lines (&#8805;80%). The described protocol allows for easy selection of several independent lines with homozygous transgene integration into the genome after UV irradiation of transgenic worms exhibiting a high rate of extrachromosomal array transmission. Furthermore, this method is quite fast and low material consuming. The possibility of rapidly generating different lines that express a particular integrated transgene is of great interest for studies focusing on gene expression pattern and regulation, protein localization, and overexpression, as well as for the development of subcellular markers.

A Rapid Protocol for Integrating Extrachromosomal Arrays With High Transmission Rate into the C. elegans Genome

This protocol describes a rapid and low material consuming procedure for the integration of transgenic extrachromosomal arrays into the Caenorhabditis elegans genome using ultra violet (UV) irradiation. Furthermore, this protocol is particularly well suited for transgenic lines that transmit extrachromosomal arrays at a high rate.

Microinjecting DNA into the cytoplasm of the syncytial gonad of Caenorhabditis elegans is the main technique used to establish transgenic lines that ...

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These age-associated disorders are characterized by the appearance of protein aggregates with fibrillary structure in the brains of these patients. Exactly why normally soluble proteins undergo an aggregation process remains poorly understood. The discovery that protein aggregation is not limited to disease processes and instead part of the normal aging process enables the study of the molecular and cellular mechanisms that regulate protein aggregation, without using ectopically expressed human disease-associated proteins. Here we describe methodologies to examine inherent protein aggregation in Caenorhabditis elegans through complementary approaches. First, we examine how to grow large numbers of age-synchronized C. elegans to obtain aged animals and we present the biochemical procedures to isolate highly-insoluble-large aggregates. In combination with a targeted genetic knockdown, it is possible to dissect the role of a gene of interest in promoting or preventing age-dependent protein aggregation by using either a comprehensive analysis with quantitative mass spectrometry or a candidate-based analysis with antibodies. These findings are then confirmed by in vivo analysis with transgenic animals expressing fluorescent-tagged aggregation-prone proteins. These methods should help clarify why certain proteins are prone to aggregate with age and ultimately how to keep these proteins fully functional.

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

The goal of the method presented here is to explore protein aggregation during normal aging in the model organism C. elegans. The protocol represents a powerful tool to study the highly insoluble large aggregates that form with age and to determine how changes in proteostasis impact protein aggregation.

方法研究线虫线虫中固有的蛋白质聚集与年龄的变化

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These ...

RNAi Feeding in Liquid Culture: A High-Throughput Method to Knockdown Gene Expression in C. elegans

Source: Groh, N. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/56464/methods-to-study-changes-inherent-protein-aggregation-with-age">Methods to Study Changes in Inherent Protein Aggregation with Age in&#160;Caenorhabditis elegans</a>.&#160;J. Vis. Exp.&#160;(2017).
RNAi is powerful genetic tool available to C. elegans researchers. This video introduces a method to treat more worms with RNAi than plate feeding by utilizing liquid culture conditions.

液体培养物中的 RNAi 补料:一种敲低秀丽隐杆线虫基因表达的高通量方法

Source: Groh, N. et al. Methods to Study Changes in Inherent Protein Aggregation with Age in&#160;Caenorhabditis elegans.&#160;J. Vis. Exp.&#160;(2017).
...

光遗传学随机突变用组蛋白miniSOG在

摘要

探索更多视频

目标

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

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

C.在体内神经元钙成像技术线虫

蠕虫病毒实验室介绍：从培养蠕虫突变

成像 C。线虫胚胎使用啶显微镜和开放源码软件

A Rapid Protocol for Integrating Extrachromosomal Arrays With High Transmission Rate into the C. elegans Genome

方法研究线虫线虫中固有的蛋白质聚集与年龄的变化

液体培养物中的 RNAi 补料:一种敲低秀丽隐杆线虫基因表达的高通量方法