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MiniSOG-mediated Photoablation in Caenorhabdtis elegans
在秀丽隐杆线虫中进行MiniSOG介导的光切除试验   

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Abstract

This protocol describes a method for light-inducible cell ablation in live worms. miniSOG (mini Singlet Oxygen Generator) generates singlet oxygen upon blue light illumination (Shu et al., 2011). Mitochondrially membrane targeted miniSOG (the first 55 a. a. of C. e. tomm-20 fused at the N’-terminus of miniSOG, termed as mito-miniSOG in the following) is transgenically expressed in specific cells/tissues (Qi et al., 2012). Groups of transgenic animals are illuminated under open field fluorescence light on a compound microscope or LED light setup for photo-ablation.

Materials and Reagents

  1. Mito-miniSOG construct* driven by your chosen promoter, e.g., Pnmr-1-mito-miniSOG (interneuron) (http://www.wormbase.org provides general information for C. e. specific promoters. http://chinook.uoregon.edu/neuron.html provides a list of neuronal promoters)
    * Requests for miniSOG construct should be addressed to Roger Y. Tsien (UC San Diego).
  2. (optional) non-green fluorescence marker to label your target cells or tissues, e.g., Pnmr-1-mCherry
  3. Control construct that is free miniSOG driven by specific promoters, e.g., Pnmr-1-miniSOG
  4. 100 mM CuCl2
  5. Filter paper
  6. NGM (nematode growth media) plates (seeded and unseeded)
  7. NGM (nematode growth  media) (see Recipes)

Equipment

  1. Fluorescence stereomicroscope (dissecting microscope, e.g., Leica, model: M165 )
  2. Upright epi-fluorescecne microscope (compound microscope, e.g., Zeiss Axio Imager 2)
  3. Prizmatix LED (UHP-Mic-LED-460), and Digital Function Generator (PASCO Scientific, catalog number: PI-9587C )
  4. Microinjection rig to generate transgenic worms

Procedure

  1. Inject your mito-miniSOG construct together with non-green fluorescence marker to produce transgenic worms.
    (Tips: Optimize the injection concentration empirically based on the promoter strength. 20-50 ng/μl would be a good starting range; for weak promoters the concentration may need to be increased up to 100 ng/μl. Screen for transgenic lines with moderate mito-miniSOG expression, i.e., the green fluorescence from mito-miniSOG should be visually detectable under a 63x objective on an epi-fluorescence microscope. If the green fluorescence can be easily detected under a 10x objective, the expression of mito-miniSOG may be too high. Mito-miniSOG transgenic animals can be kept in standard incubators under the cover of aluminum foil, and caution should be taken in keeping animals away from continuous room light exposure).
  2. L4 (or any developmental stages) transgenic animals are isolated using behaviorally visible markers or non-GFP co-injection markers under fluorescence dissecting microscope; avoid exposure to blue light.
  3. If you intend to use compound microscope for blue light illumination, precede to steps 4-10. If a LED light is used, go to steps 11-13.
  4. Cut a filter paper in a ring shape with the outer diameter of 50 mm (to fit into the 60 mm dish) and the inner diameter of 15 mm, about the size of the illumination spot. Place the filter paper on an unseeded 60 mm NGM plate.
  5. Drop ample amount of 100 mM CuCl2 solution (about 0.5 ml) onto the filter paper until the paper is adequately wet; the Cu2+ is to restrict worms inside the illumination spot, as worms show aversion to Cu2+.
  6. Add OP50 paste in the ring opening by pressing a chunk of seeded agar (from a seeded plate) upside down then flipping it off. Addition of OP50 is to keep animals well fed during illumination.
  7. Transfer transgenic worms inside the copper ring.
  8. Place the plate without cover on the compound microscope stage right under one empty position of the turret (or remove one objective lens), such that blue light passes down directly onto the plate.
  9. Turn on the blue fluorescence light (475 ± 20 nm). An example of the light intensity worms received was 57 mW/cm2 on compound microscope.
    (Tips: Animals can be exposed to either continuous blue light, or pulse blue light (0.5 sec on, 1.5 sec off). The pulse light is provided by an electrical shutter, e.g., 30 mm W/HS, LUDL Electronics and a signal device, e.g., Uniblitz unit (VMM-D1)).
  10. Illuminate animals for 30 min to 1 h; optimal length should be determined empirically with respect to the specific microscopes and light sources and the transgenes. After light illumination, worms were transferred to freshly seeded plates prior to any analysis.
  11. (To use LED for illumination) Drop about 0.5-1 ml of 100 mM CuCl2 solution by the edge of an 40 mm NGM plate (40 mm is about the size of the illumination spot under our LED setup) and swirl the liquid around the dish wall for a couple of times, then remove excessive liquid.
  12. Transfer transgenic worms onto the center of the 40 mm NGM plate (we prefer to use seeded plates unless other specific conditions are demanded otherwise). Place the plates under the LED light source. The distance between worms and the LED light plate is about 12 cm. Turn on the LED light. The frequency of LED light is set to 4 Hz. The intensity of blue light (460 nm) from LED set up is around 800 mW/cm2.
  13. Illuminate the worms for any desired length of time. Then transfer worms onto a new seeded plate.
    (Tips: Optimal exposure time should be determined empirically, depending on cell type and expression levels of mito-miniSOG. For example, flashes 4 times per sec for 2 min can sufficiently damage hypodermis expressing mito-miniSOG, but much longer time illumination would be needed to kill neurons.)

Recipes

  1. NGM (nematode growth media)

Acknowledgments

This protocol is adapted from Qi et al. (2012) and Shu et al. (2011).

References

  1. Qi, Y. B., Garren, E. J., Shu, X., Tsien, R. Y. and Jin, Y. (2012). 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.
  2. Shu, X., Lev-Ram, V., Deerinck, T. J., Qi, Y., Ramko, E. B., Davidson, M. W., Jin, Y., Ellisman, M. H. and Tsien, R. Y. (2011). A genetically encoded tag for correlated light and electron microscopy of intact cells, tissues, and organisms. PLoS Biol 9(4): e1001041.

简介

该协议描述了在活蠕虫中的光诱导细胞消融的方法。 miniSOG(微型单态氧发生器)在蓝光照射时产生单线态氧(Shu等人,2011)。 线粒体膜靶向的miniSOG(在miniSOG的N'末端融合的Cys的第一个55aa,在下文中称为mito-miniSOG)在特定细胞/组织中转基因表达, 组织(Qi等人,2012)。 在复合显微镜或用于光消融的LED光设置下在开放场荧光下照射转基因动物组。

材料和试剂

  1. 由所选启动子驱动的Mito-miniSOG构建体,例如, Pnmr-1-mito-mini SOG (interneuron)( http://www.wormbase.org 提供了 C。e。特定启动子的一般信息。 a href ="http://chinook.uoregon.edu/neuron.html"target ="_blank"> http://chinook.uoregon.edu/neuron.html 提供了神经元启动子列表)
    *请求miniSOG构建应该提交给Roger Y. Tsien(加利福尼亚州圣地亚哥)。
  2. (可选)非绿色荧光标记物标记目标细胞或组织,例如 , Pnmr-1-mCherry
  3. 由特定启动子驱动的免费miniSOG的控制构建体,例如 , Pnmr-1-miniSOG
  4. 100mM CuCl 2 v/v
  5. 过滤纸
  6. NGM(线虫生长培养基)平板(种子和未种子)
  7. NGM(线虫生长介质)(参见食谱)

设备

  1. 荧光立体显微镜(解剖显微镜,例如,Leica,型号:M165)
  2. 直立表面荧光显微镜(复合显微镜,例如Zeiss Axio Imager 2)
  3. Prizmatix LED(UHP-Mic-LED-460)和数字函数发生器(PASCO Scientific,目录号:PI-9587C)
  4. 显微注射钻机生成转基因蠕虫

程序

  1. 注射您的mito-miniSOG结构与非绿色荧光标记,以产生转基因蠕虫 (提示:基于启动子强度,根据经验优化注射浓度20-50ng /μl是良好的起始范围;对于弱启动子,浓度可能需要增加至100ng /μl 。对具有中等mito-miniSOG表达的转基因品系的筛选,即来自mito-miniSOG的绿色荧光应当在epi荧光显微镜的63x物镜下可目测检测,如果绿色荧光可以容易地在10x目标下检测到的,mito-miniSOG的表达可能太高。可以将Mito-miniSOG转基因动物保存在标准孵化器中的铝箔覆盖下,并且应当小心保持动物远离连续室内光照) 。
  2. 使用行为可见的标记或非GFP共注射标记在荧光解剖显微镜下分离L4(或任何发育阶段)转基因动物;避免暴露于蓝光。
  3. 如果您打算使用复合显微镜进行蓝光照明,请先执行步骤4-10。如果使用LED灯,请转到步骤11-13。
  4. 将外径为50mm(以适应60mm的盘)和内径为15mm的环形滤纸切成约照射点的尺寸。将滤纸放在未涂布的60 mm NGM板上。
  5. 将足量的100mM CuCl 2溶液(约0.5ml)滴到滤纸上,直到纸充分润湿; Cu 2 + 是限制照明点内的蠕虫,因为蠕虫显示对Cu 2 + 的厌恶。
  6. 通过按下一块种子琼脂(从种子板)颠倒,然后翻转它添加OP50糊在环开口。添加OP50是为了在照明期间保持动物良好的喂养
  7. 在铜环内转移转基因蠕虫。
  8. 将无盖板放在复合显微镜载物台上转盘正下方一个空位置(或取下一个物镜),使蓝光直接通过板。
  9. 打开蓝色荧光(475±20 nm)。在复合显微镜上接收到的光强度蠕虫的实例为57mW/cm 2 。
    (提示:动物可以暴露于连续的蓝光或脉冲蓝光(0.5秒开,1.5秒关闭)。脉冲光由电子快门提供, 30mm W/HS,LUDL Electronics和信号装置,例如,Uniblitz单元(VMM-D1))。
  10. 照亮动物30分钟至1小时;最佳长度应根据特定显微镜和光源以及转基因经验确定。光照后,在进行任何分析之前,将蠕虫转移到新接种的平板上
  11. (为了使用LED照明)通过40mm NGM板的边缘(40mm约为我们的LED设置下的照明斑点的尺寸)滴下约0.5-1ml的100mM CuCl 2溶液)并使盘壁周围的液体旋转几次,然后除去过量的液体。
  12. 转移转基因蠕虫到40毫米NGM板的中心(我们喜欢使用种板,除非另有具体条件,否则要求)。将板放在LED光源下。蜗杆与LED灯板之间的距离约为12厘米。打开LED灯。 LED灯的频率设置为4 Hz。来自LED装置的蓝光(460nm)的强度为约800mW/cm 2 。
  13. 照亮蠕虫任何所需的时间长度。 然后将蠕虫转移到新的接种板上 (提示:根据细胞类型和mito-miniSOG的表达水平,根据经验确定最佳暴露时间。例如,每秒闪烁4次,持续2分钟可充分损害表达hypodermis的mito-miniSOG, 但是需要更长时间的照明来杀死神经元。)

食谱

  1. NGM(线虫生长介质)

致谢

该协议改编自Qi等人(2012)和Shu等人(2011)。

参考文献

  1. Qi,Y. B.,Garren,E.J.,Shu,X.,Tsien,R.Y.and Jin,Y。(2012)。 Caenorhabditis elegans中的光诱导细胞消融使用遗传编码的单线态氧 生成蛋白质miniSOG。 Proc Natl Acad Sci USA 109(19):7499-7504。
  2. Shu,X.,Lev-Ram,V.,Deerinck,T.J.,Qi,Y.,Ramko,E.B.,Davidson,M.W.,Jin,Y.,Ellisman,M.H.and Tsien, 用于完整细胞,组织和生物的相关光和电子显微镜的遗传编码标签。 a> PLoS Biol 9(4):e1001041。
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Qi, Y. and Xu, S. (2013). MiniSOG-mediated Photoablation in Caenorhabdtis elegans. Bio-protocol 3(1): e316. DOI: 10.21769/BioProtoc.316.
  2. Qi, Y. B., Garren, E. J., Shu, X., Tsien,R. Y. and Jin, Y. (2012). Photo-inducible cell ablation in Caenorhabditiselegans using the genetically encoded singlet oxygen generating proteinminiSOG. Proc Natl Acad Sci U S A 109(19): 7499-7504.
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