光遗传学随机突变用组蛋白miniSOG在<em>℃。线虫</em
Summary
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。
Abstract
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.
Introduction
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。
Protocol
Representative Results
Discussion
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?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
The work is supported by HHMI. We thank our lab members for their help with testing the protocol and revising the manuscript.
Materials
| Ultra High Power LED light source | Prizmatix | UHP-mic-LED-460 | 460 ± 5 nm |
| LED controller | Prizmatix | UHPLCC-01 | |
| Digital function generator/amplifier | PASCO | PI-9587C | PI-9587C is no longer available. The replacement is PI-8127. |
| BNC cable male/male | THORLABS | CA3136 | |
| USB-TTL interface | Prizmatix | Optional | |
| Photometer | THORLABS | PM50 and Model D10MM | |
| Filter paper | Whatman | 1001-110 | |
| Copper chloride dihydrate (CuCl2∙2H2O) | Sigma | C6641 | |
| Stereomicroscope | Leica | MZ95 | |
| NGM plate | Dissolve 5g NaCl, 2.5g Peptone, 20g Agar, 10 µg/ml cholesterol in 1 L H2O. After autoclaving, add 1 mM CaCl2, 1 mM MgSO4, 25 mM KH2PO4(pH6.0). | ||
| Lysis solution | 10 mM Tris(pH8.8), 50 mM KCl, 0.1% Triton X100, 2.5 mM MgCl2, 100 µg/ml Proteinase K |
Riferimenti
- Jorgensen, E. M., Mango, S. E. The art and design of genetic screens: caenorhabditis elegans. Nat Rev Genet. 3 (5), 356-369 (2002).
- Kutscher, L. M., Shaham, S. Forward and reverse mutagenesis in C. elegans. WormBook. , 1-26 (2014).
- Collins, J., Saari, B., Anderson, P. Activation of a transposable element in the germ line but not the soma of Caenorhabditis elegans. Nature. 328 (6132), 726-728 (1987).
- Noma, K., Jin, Y. Optogenetic mutagenesis in Caenorhabditis elegans. Nat Commun. 6, 8868 (2015).
- Cooke, M. S., Evans, M. D., Dizdaroglu, M., Lunec, J. Oxidative DNA damage: mechanisms, mutation, and disease. FASEB J. 17 (10), 1195-1214 (2003).
- Shu, X., et al. A genetically encoded tag for correlated light and electron microscopy of intact cells, tissues, and organisms. PLoS Biol. 9 (4), 1001041 (2011).
- Ruiz-Gonzalez, R., et al. Singlet oxygen generation by the genetically encoded tag miniSOG. J Am Chem Soc. 135 (26), 9564-9567 (2013).
- Pimenta, F. M., Jensen, R. L., Breitenbach, T., Etzerodt, M., Ogilby, P. R. Oxygen-dependent photochemistry and photophysics of “miniSOG,” a protein-encased flavin. Photochem Photobiol. 89 (5), 1116-1126 (2013).
- Frokjaer-Jensen, C., et al. Random and targeted transgene insertion in Caenorhabditis elegans using a modified Mos1 transposon. Nat Methods. 11 (5), 529-534 (2014).
- Brenner, S. The genetics of Caenorhabditis elegans. Genetica. 77 (1), 71-94 (1974).
- Chaudhuri, J., Parihar, M., Pires-daSilva, A. An introduction to worm lab: from culturing worms to mutagenesis. J Vis Exp. (47), (2011).
- Berkowitz, L. A., Knight, A. L., Caldwell, G. A., Caldwell, K. A. Generation of stable transgenic C. elegans using microinjection. J Vis Exp. (18), (2008).
- Mello, C., Fire, A. DNA transformation. Methods Cell Biol. 48, 451-482 (1995).
- Fay, D., Bender, A. Genetic mapping and manipulation: chapter 4–SNPs: introduction and two-point mapping. WormBook. , 1-7 (2006).
- Lee, P. Y., Costumbrado, J., Hsu, C. Y., Kim, Y. H. Agarose gel electrophoresis for the separation of DNA fragments. J Vis Exp. (62), (2012).
- Westberg, M., Holmegaard, L., Pimenta, F. M., Etzerodt, M., Ogilby, P. R. Rational design of an efficient, genetically encodable, protein-encased singlet oxygen photosensitizer. J Am Chem Soc. 137 (4), 1632-1642 (2015).
- Xu, S., Chisholm, A. D. Highly efficient optogenetic cell ablation in C. elegans using membrane-targeted miniSOG. Sci Rep. 6, 21271 (2016).
- Mariol, M. C., Walter, L., Bellemin, S., Gieseler, K. A rapid protocol for integrating extrachromosomal arrays with high transmission rate into the C. elegans genome. J Vis Exp. (82), (2013).
- Lin, J. Y., et al. Optogenetic inhibition of synaptic release with chromophore-assisted light inactivation (CALI). Neuron. 79 (2), 241-253 (2013).
- Minevich, G., Park, D. S., Blankenberg, D., Poole, R. J., Hobert, O. CloudMap: A Cloud-Based Pipeline for Analysis of Mutant Genome Sequences. Genetica. 192 (>4), 1249-1269 (2012).
- Qi, Y., Xu, S. MiniSOG-mediated Photoablation in Caenorhabdtis elegans. Bio-protocol. 3 (1), 316 (2013).
- Qi, Y. B., Garren, E. J., Shu, X., Tsien, R. Y., Jin, Y. Photo-inducible cell ablation in Caenorhabditis elegans using the genetically encoded singlet oxygen generating protein miniSOG. Proc Natl Acad Sci U S A. 109 (19), 7499-7504 (2012).
- Nagel, G., et al. Light activation of channelrhodopsin-2 in excitable cells of Caenorhabditis elegans triggers rapid behavioral responses. Curr Biol. 15 (24), 2279-2284 (2005).
