Transgenic Pigs with Red-Shifted Channelrhodopsin-Citrine Fusion Proteins

具有红移通道视紫红质-黄水晶融合蛋白的转基因猪

• 批准号: 10065184
• 负责人: Leah R Reznikov
• 金额: $ 46.22万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-01 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10065184
• 关键词: Address; Anatomy; Animal Model; Animals; Area; Back; Basic Science; Benchmarking; Biomedical Research; Calmodulin; Cells; Chimeric Proteins; Choline O-Acetyltransferase; Communicable Diseases; Complex; Databases; Device or Instrument Development; Disease; Doctor of Philosophy; Elements; Embryo; Event; Family suidae; Female; Fertility; Fibroblasts; Florida; Genetic; Genome; Goals; Green Fluorescent Proteins; Housing; Human; Image; Laboratories; Life; Light; Medicine; Methods; Missouri; Modeling; Monitor; Nervous system structure; Neuraxis; Neurons; Neurosciences; Nuclear; Organ; Paste substance; Peripheral Nerves; Peripheral Nervous System; Phototoxicity; Physiology; Play; Process; Proteins; Records; Research; Research Personnel; Resources; Rodent; Rodent Model; Role; Skin; Synapsins; System; Technology; Therapeutic; Therapeutic Intervention; Transgenic Organisms; Translations; Treatment-related toxicity; Universities; Variant; Visualization; Work; bench to bedside; blastocyst; cholinergic neuron; cost; fetal; male; nerve supply; new technology; promoter; relating to nervous system; sensor; somatic cell nuclear transfer; technology development; tool; transgene expression

PROJECT SUMMARY Large animals are increasingly utilized in biomedical research and offer an advantage in that their anatomy and physiology closely parallel humans. Yet despite these advantages, rodent models dominate many areas of research, including neuroscience. This might be due in part to the greater number of technologies available to rodent researchers. In the current application, the investigator proposes to create a new technology to facilitate large animal researchers and bridge this gap. Specifically, she proposes to create transgenic pigs with red-shifted channelrhodopsin-citrine fusion proteins or green fluorescent protein/calmodulin protein sensors expressed in neurons. This will allow for simultaneous visualization of neurons (and their innervation), and the ability to precisely control or monitor neural activity. An important advantage of the red-shifted channelrhodopsin variant is that it can be activated by near far red light (~630 nm), thus decreasing phototoxic events and opening the door to less-invasive methods of neural activation (i.e. through the skin). Moreover, the fusion of the green fluorescent protein derivative, citrine, will allow for identification of specific neural elements expressing channelrhodpsin, as well as enable visualization of organ innervation. To create the transgenic pigs, the investigator proposes to target porcine fetal fibroblasts using piggyBac transposon technology. The piggyBac transposon system allows for “cut and paste” integration of the targeting construct into the porcine genome. An advantage of this approach is that it promotes stable transgene expression. Transgenic pigs will be derived from the targeted fetal fibroblasts at the University of Missouri where Dr. Randall Prather and his team will perform somatic cell nuclear transfer. Embryos containing the targeted nuclear material will be transferred to a surrogate gilt and allowed to develop until term. Transgenic pigs will then be studied and characterized in the investigator's lab at the University of Florida. The proposed work is completely aligned with the priorities of SPARC and will facilitate the imaging and targeting of peripheral nerves with end organs in a large animal model. Moreover, its utility is not limited to peripheral nervous system researchers, as central nervous system neurons will also express channelrhodopsin-citrine fusion proteins or green fluorescent protein/calmodulin protein sensors. Thus, the work proposed in this application has the potential to greatly accelerate the neuroscience field and propel the bench-to-bedside process.

项目总结 大型动物越来越多地被用于生物医学研究，并提供了它们的优势 解剖学和生理学与人类极为相似。然而，尽管有这些优势，啮齿动物模型 在许多研究领域占据主导地位，包括神经科学。这可能在一定程度上是由于 啮齿动物研究人员可用的技术数量。在当前的应用程序中，调查员 建议创造一种新技术，为大型动物研究人员提供便利，并弥合这一差距。 具体地说，她建议创造红移通道视紫红质-黄嘌呤融合的转基因猪 在神经元中表达的蛋白质或绿色荧光蛋白/钙调素蛋白传感器。这将允许 用于同时可视化神经元(及其神经)，以及精确控制或 监测神经活动。红移通道视紫红质变体的一个重要优势是它 可被近红光(~630 nm)激活，从而减少光毒事件并打开 通向侵入性较小的神经激活方法(即通过皮肤)。此外，两国的融合 绿色荧光蛋白的衍生物，柠檬碱，将允许识别特定的神经元素 还可以显示器官的神经支配情况。要创建 在转基因猪中，研究人员建议使用iggyBac来靶向猪胎儿成纤维细胞 转座子技术。该系统允许“剪切和粘贴”整合的 将其定向构建到猪基因组中。这种方法的一个优点是它促进了稳定 转基因表达。转基因猪将从目标胎儿成纤维细胞中衍生出来 密苏里大学兰德尔·普拉瑟博士和他的团队将在那里进行体细胞核 调职。含有目标核材料的胚胎将被移植到代理金边，并 允许发育到学期。然后将对转基因猪进行研究，并在 佛罗里达大学的调查员实验室。拟议的工作完全符合 SPARC的优先事项，并将促进周围神经与末端器官的成像和靶向 在一个大型动物模型中。而且，它的用途并不局限于周围神经系统的研究人员， 作为中枢神经系统，神经元也会表达通道视紫红质-柠檬碱融合蛋白或绿色 荧光蛋白/钙调素蛋白传感器。因此，本申请中提议的工作具有 这将极大地加速神经科学领域的发展，并推动从病床到床边的进程。