斑马鱼早期胚胎和肿瘤细胞电活动的可视化研究
Summary
在这里, 我们展示了创建一个细胞电压报告斑马鱼线的过程, 以可视化胚胎发育, 运动和鱼类肿瘤细胞在体内。
Abstract
生物电, 由离子通道和细胞膜上的泵介导的内源电信号, 在兴奋性神经细胞和肌肉细胞的信号过程和其他许多生物学过程中起着重要作用, 如胚胎发展模式。但是, 在脊椎动物胚胎发生中需要进行体内电活动监测。基因编码的荧光电压指示器 (GEVIs) 的发展, 使我们有可能为这一挑战提供解决方案。在这里, 我们描述如何创建一个转基因的电压指示器斑马鱼使用已建立的电压指示器, ASAP1 (动作电位加速传感器 1), 作为一个例子。本研究选择了 Tol2 套件和无处不在的斑马鱼启动子ubi 。我们还解释了网关站点特定克隆的过程, Tol2 座子斑马鱼转基因, 以及早期的鱼类胚胎和鱼类肿瘤的成像过程, 使用常规 epifluorescent 显微镜。利用这条鱼线, 我们发现在斑马鱼胚胎发生和鱼类幼虫运动过程中存在着细胞电压的变化。此外, 据观察, 在一些斑马鱼恶性周围神经鞘肿瘤, 肿瘤细胞一般极化相比, 周围正常组织。
Introduction
生物电是指由离子通道和位于细胞膜1上的泵所介导的内源电子信号。细胞膜上的离子交换, 以及耦合电位和电流的变化, 对于兴奋性神经细胞和肌肉细胞的信号过程是必不可少的。此外, 生物电和离子梯度也有多种其他重要的生物学功能, 包括能量储存, 生物合成和代谢物运输。生物电信号也被发现作为胚胎模式形成的调节器, 如体轴, 细胞周期和细胞分化的1。因此, 这是至关重要的了解许多人类先天性疾病的结果, 这类信号的错误监管。虽然膜片钳已被广泛用于记录单个细胞, 但它仍然远未理想的同时监测在胚胎发育过程中的多个细胞在体内。此外, 由于其特异性、敏感性和毒性, 电压敏感小分子对于体内应用也不理想。
创建各种基因编码的荧光电压指示器 (GEVIs) 提供了一个新的机制来克服这一问题, 并允许易于应用研究胚胎发育, 即使他们最初是为了监测神经单元格2,3。当前可用的 GEVIs 之一是动作电位 1 (ASAP1)4的加速传感器。它由电压敏感磷酸酶的电压传感域和循环排列绿色荧光蛋白组成。因此, ASAP1 允许可视化的细胞电位变化 (极化: 明亮的绿色; 退极化: 深绿色)。ASAP1 有2毫秒的动力学, 可以跟踪阈的潜在变化4。因此, 这一遗传工具允许在实时生物监测活细胞的新水平的有效性。进一步了解生物电在胚胎发育和许多人类疾病 (如癌症) 中的作用, 将为疾病治疗和预防的关键机制提供新的基础。
斑马鱼已被证明是一个强大的动物模型, 研究发育生物学和人类疾病, 包括癌症5,6。他们分享70% 同源基因与人, 并且他们有相似的脊椎动物生物7。斑马鱼提供相对容易的护理, 大的离合器大小的鸡蛋, 易于处理的遗传学, 容易转基因, 和透明的外部胚胎发育, 这使他们成为一个优越的系统在体内成像5,6。随着大量的突变鱼线来源已经存在和一个完全有序的基因组, 斑马鱼将提供一个相对无限的科学发现范围。
为了研究细胞的体内实时电活动, 我们利用斑马鱼模型系统和 ASAP1。本文介绍了如何利用 Tol2 座子转基因将荧光电压生物传感器 ASAP1 到斑马鱼基因组中, 并在胚胎发育、鱼类幼虫运动和活肿瘤中可视化细胞电活动.
Protocol
斑马鱼被安置在一个 AAALAC 批准的动物设施中, 所有的实验都是根据普渡动物保育和使用委员会 (PACUC) 批准的协议进行的。 1. Tol2 座子质粒结构制备 注: Tol2 是在鳉鱼中发现的座子, 广泛应用于斑马鱼研究社区8,9。它已成功地应用于网关站点特定的基于重组的克隆系统, 并被称为 Tol2 套件10。Tol2 套…
Representative Results
在一个成功的注射, 超过50% 注入鱼类胚胎将显示一定程度的绿色荧光在体细胞, 其中大部分将是积极的 Tol2 座子的消费检测 (图 2)。经过2-4 代与 wildtype 鱼的杂交 (直到荧光鱼达到 50%, 预期的孟德尔比), 转基因鱼被用于成像实验, 以跟踪细胞膜电位在胚胎发育过程中。首先, 在斑马鱼早期胚胎发育阶段, 在整个细胞周期内检查膜电位的变化。据观察, 细胞…
Discussion
虽然早在胚胎发育和人类疾病中发现了细胞和组织水平的电活动, 但体内动态电变化及其生物学作用仍然很大程度上是未知的。其中一个主要的挑战是可视化和量化的电气变化。膜片钳技术是跟踪单细胞的一种突破, 但是它在脊椎动物胚胎中的应用是有限的, 因为它们是由许多细胞组成的。由于敏感度、特异度和毒性, 目前的化学电压染料也不理想。最近在 GEVIs 发明方面的努力为我们提供了?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
本出版物所报道的研究工作得到国家卫生研究院全国普通医学研究所的支持, R35GM124913、普渡大学 PI4D 奖励计划和 PVM 内部竞争基础研究基金项目。内容完全是作者的责任, 不一定代表供资代理人的官方意见。我们感谢高一川上刚为 Tol2 构造, 林为 ASAP1 构造, 和伦纳德 Zon为ubi 启动子构造通过 Addgene。
Materials
| 14mL cell culture tubes | VWR | 60818-725 | E.Coli culture |
| Agarose electrophoresis tank | Thermo Scientific | Owl B2 | DNA eletrophoresis |
| Agarose RA | Amresco | N605-500G | For making the injection gels |
| Attb1-ASAP1-F primer | IDT DNA | GGGGACAAGTTTGTACAAAAAAGCAGGCTTCACCATGGAGACGACTGTGAGGTATGAACA | ASAP1 coding region amplification for subcloning |
| Attb2-ASAP1-R primer | IDT DNA | GGGGACCACTTTGTACAAGAAAGCTGGGTCTTAGGTTACCACTTCAAGTTGTTTCTTCTGTGAAGCCA | ASAP1 coding region amplification for subcloning |
| Bright field dissection scope | Nikon | SMZ 745 | Dechorionation, microinjection, mounting |
| Color camera | Zeiss | AxioCam MRc | Fish embryo image recording |
| Concave slide | VWR | 48336-001 | For holding fish embryos during imaging process |
| Disposable transfer pipette 3.4 ml | Thermo Scientific | 13-711-9AM | Fish embryos and water transfer |
| Endonuclease enzyme, Not I | NEB | R0189L | For linearizing plasmid DNA |
| Epifuorescent compound scope | Zeiss | Axio Imager.A2 | Fish embryo imaging |
| Epifuorescent stereo dissection scope | Zeiss | Stereo Discovery.V12 | Fish embryo imaging |
| Fluorescent light source | Lumen dynamics | X-cite seris 120 | Light source for fluorescence microscopes |
| Forceps #5 | WPI | 500342 | Dechorionation and needle breaking |
| Gateway BP Clonase II Enzyme mix | Thermo Scientific | 11789020 | Gateway BP recombination cloning |
| Gateway LR Clonase II Plus enzyme | Thermo Scientific | 12538120 | Gateway LR recombination cloning |
| Gel DNA Recovery Kit | Zymo Research | D4002 | DNA gel purification |
| Loading tip | Eppendorf | 930001007 | For loading injection solution into capilary needles |
| Methylcellulose (1600cPs) | Alfa Aesar | 43146 | Fish embryo mounting |
| Methylene blue | Sigma-Aldrich | M9140 | Suppresses fungal outbreaks in Petri dishes |
| Microinjection mold | Adaptive Science Tools | TU-1 | To prepare agaorse mold tray for holding fish embryos during injection |
| Microinjector | WPI | Pneumatic Picopump PV820 | Microinjection injector |
| Micro-manipulator | WPI | Microinjector mm33 rechts | Microinjection operation |
| Micropipette puller | Sutter instrument | P-1000 | For preparing capillary needle |
| Mineral oil | Amresco | J217-500ml | For calibrating injection volume |
| mMESSAGE mMACHINE SP6 Transcription Kit | Thermo Scientific | AM1340 | mRNA in vitro transcription |
| Monocolor camera | Zeiss | AxioCam MRm | Fish embryo image recording |
| Plasmid Miniprep Kit | Zymo Research | D4020 | Prepare small amount of plasmid DNA |
| Plastic Petri dishes | VWR | 25384-088 | For holding fish or fish embryos during imaging process |
| RNA Clean & Concentrator-5 | Zymo Research | R1015 | mRNA cleaning after in vitro transcription |
| Spectrophotometer | Thermo Scientific | NanoDrop 2000 | For measuring DNA and RNA concentrations |
| Stage Micrometer | Am Scope | MR100 | Microinjection volume calibration |
| Thermocycler | Bio-Rad | T100 | DNA amplification for gene cloning |
| Thin wall glass capillaries | WPI | TW100F-4 | Raw glass for making cappilary needle |
| Tol2-exL1 primer | IDT DNA | GCACAACACCAGAAATGCCCTC | Tol2 excise assay |
| Tol2-exR primer | IDT DNA | ACCCTCACTAAAGGGAACAAAAG | Tol2 excise assay |
| TOP10 Chemically Competent E. coli | Thermo Scientific | C404006 | Used for transformation during gene cloning |
| Tricaine mesylate | Sigma-Aldrich | A5040 | For anesthetizing fish or fish embryos |
| UV trans-illuminator 302nm | UVP | M-20V | DNA visualization |
| Water bath | Thermo Scientific | 2853 | For transformation process of gene cloning |
References
- Levin, M. Molecular bioelectricity: how endogenous voltage potentials control cell behavior and instruct pattern regulation in vivo. Molecular Biology of the Cell. 25 (24), 3835-3850 (2014).
- Storace, D., et al. Toward Better Genetically Encoded Sensors of Membrane Potential. Trends in Neuroscience. 39 (5), 277-289 (2016).
- Inagaki, S., Nagai, T. Current progress in genetically encoded voltage indicators for neural activity recording. Current Opinion in Chemical Biology. 33, 95-100 (2016).
- St-Pierre, F., et al. High-fidelity optical reporting of neuronal electrical activity with an ultrafast fluorescent voltage sensor. Nature Neuroscience. 17 (6), 884-889 (2014).
- Lieschke, G. J., Currie, P. D. Animal models of human disease: zebrafish swim into view. Nature Reviews Genetics. 8 (5), 353-367 (2007).
- Santoriello, C., Zon, L. I. Hooked! Modeling human disease in zebrafish. Journal of Clinical Investigation. 122 (7), 2337-2343 (2012).
- Howe, K., et al. The zebrafish reference genome sequence and its relationship to the human genome. Nature. , (2013).
- Kawakami, K., Shima, A., Kawakami, N. Identification of a functional transposase of the Tol2 element, an Ac-like element from the Japanese medaka fish, and its transposition in the zebrafish germ lineage. Proceedings of the National Academy of Science USA. 97 (21), 11403-11408 (2000).
- Urasaki, A., Asakawa, K., Kawakami, K. Efficient transposition of the Tol2 transposable element from a single-copy donor in zebrafish. Proceedings of the National Academy of Science USA. 105 (50), 19827-19832 (2008).
- Kwan, K. M., et al. The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs. Developmental Dynamics. 236 (11), 3088-3099 (2007).
- Mosimann, C., et al. Ubiquitous transgene expression and Cre-based recombination driven by the ubiquitin promoter in zebrafish. Development. 138 (1), 169-177 (2011).
- Lorenz, T. C. Polymerase chain reaction: basic protocol plus troubleshooting and optimization strategies. Journal of Visualized Experiments. (63), e3998 (2012).
- Ordovas, J. M. Separation of small-size DNA fragments using agarose gel electrophoresis. Methods in Molecular Biology. 110, 35-42 (1998).
- Downey, N. Extraction of DNA from agarose gels. Methods Mol Biol. 235, 137-139 (2003).
- Desjardins, P., Conklin, D. NanoDrop microvolume quantitation of nucleic acids. Journal of Visualized Experiments. 45 (45), (2010).
- Green, M. R., Sambrook, J. . Molecular cloning : a laboratory manual. , (2012).
- Zhang, S., Cahalan, M. D. Purifying plasmid DNA from bacterial colonies using the QIAGEN Miniprep Kit. Journal of Visualized Experiments. (6), 247 (2007).
- Meeker, N. D., Hutchinson, S. A., Ho, L., Trede, N. S. Method for isolation of PCR-ready genomic DNA from zebrafish tissues. Biotechniques. 43 (5), (2007).
- Kawakami, K., Koga, A., Hori, H., Shima, A. Excision of the tol2 transposable element of the medaka fish, Oryzias latipes, in zebrafish, Danio rerio. Gene. 225 (1-2), 17-22 (1998).
- Westerfield, M. . The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio). , (2000).
- Amsterdam, A., et al. Many ribosomal protein genes are cancer genes in zebrafish. PLoS Biology. 2 (5), E139 (2004).
- Lai, K., et al. Many ribosomal protein mutations are associated with growth impairment and tumor predisposition in zebrafish. Developmental Dynamics. 238 (1), 76-85 (2009).
- Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. Stages of embryonic development of the zebrafish. Developmental Dynamics. 203 (3), 253-310 (1995).
- Zhang, G., et al. Comparative oncogenomic analysis of copy number alterations in human and zebrafish tumors enables cancer driver discovery. PLoS Genetics. 9 (8), e1003734 (2013).
- Zhang, G., et al. Highly aneuploid zebrafish malignant peripheral nerve sheath tumors have genetic alterations similar to human cancers. Proceedings of the National Academy of Science USA. 107 (39), 16940-16945 (2010).
- Urrego, D., Tomczak, A. P., Zahed, F., Stuhmer, W., Pardo, L. A. Potassium channels in cell cycle and cell proliferation. Philosophical Transactions of the Royal Society of London Series B. 369 (1638), 20130094 (2014).
- Yang, H. H., et al. Subcellular Imaging of Voltage and Calcium Signals Reveals Neural Processing In Vivo. Cell. 166 (1), 245-257 (2016).
- Chamberland, S., et al. Fast two-photon imaging of subcellular voltage dynamics in neuronal tissue with genetically encoded indicators. Elife. 6, (2017).
- Hochbaum, D. R., et al. All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins. Nature Methods. 11 (8), 825-833 (2014).
- Sugiyama, M., et al. Illuminating cell-cycle progression in the developing zebrafish embryo. Proceedings of the National Academy of Science USA. 106 (49), 20812-20817 (2009).
Cite This Article
Silic, M. R., Zhang, G. Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors. J. Vis. Exp. (134), e57330, doi:10.3791/57330 (2018).