Improved Long-Term Imaging of Embryos with Genetically Encoded α-Bungarotoxin.
Improved Long-Term Imaging of Embryos with Genetically Encoded α-Bungarotoxin.
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DOI：
10.1371/journal.pone.0134005
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Megason SG
中科院分区：
文献类型：
--
作者：
Swinburne IA;Mosaliganti KR;Green AA;Megason SG
显微镜和遗传标记策略的快速发展为胚胎发育的延时成像创造了新的机会。然而，长期固定胚胎同时保持正常发育的方法几乎没有变化。在斑马鱼中，目前的固定技术依赖于麻醉剂三卡因。不幸的是，在足够高的浓度下长时间的三卡因治疗会对胚胎的发育产生不良的副作用。我们评估了三种替代的固定策略：组合浸泡在三卡因和异丁香酚中，注射α-银环蛇毒素蛋白，和注射α-银环蛇毒素mRNA。我们发现的证据之间的合作tricaine和异丁香酚，以改善健康的不动性。然而，即使结合这些麻醉剂也会对长期发育产生负面影响。α-银环蛇毒素是一种来自蛇毒的小蛋白质，可不可逆地结合并灭活乙酰胆碱受体。我们发现，α-银环蛇毒素无论是从蛇中纯化的蛋白质，还是从密码子优化的合成基因在斑马鱼中内源性表达的蛋白质，都可以使胚胎长时间存活，而对健康的影响或发育延迟很少。使用α-银环蛇毒素mRNA注射，我们获得了从1-细胞期到受精后3天的斑马鱼胚胎发生的完整电影，正常健康，没有抽搐。这些结果表明，内源性表达的α-银环蛇毒素为延时显微镜提供了前所未有的固定性和健康。
Rapid advances in microscopy and genetic labeling strategies have created new opportunities for time-lapse imaging of embryonic development. However, methods for immobilizing embryos for long periods while maintaining normal development have changed little. In zebrafish, current immobilization techniques rely on the anesthetic tricaine. Unfortunately, prolonged tricaine treatment at concentrations high enough to immobilize the embryo produces undesirable side effects on development. We evaluate three alternative immobilization strategies: combinatorial soaking in tricaine and isoeugenol, injection of α-bungarotoxin protein, and injection of α-bungarotoxin mRNA. We find evidence for co-operation between tricaine and isoeugenol to give immobility with improved health. However, even in combination these anesthetics negatively affect long-term development. α-bungarotoxin is a small protein from snake venom that irreversibly binds and inactivates acetylcholine receptors. We find that α-bungarotoxin either as purified protein from snakes or endogenously expressed in zebrafish from a codon-optimized synthetic gene can immobilize embryos for extended periods of time with few health effects or developmental delays. Using α-bungarotoxin mRNA injection we obtain complete movies of zebrafish embryogenesis from the 1-cell stage to 3 days post fertilization, with normal health and no twitching. These results demonstrate that endogenously expressed α-bungarotoxin provides unprecedented immobility and health for time-lapse microscopy.