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.

项目概要 大型动物越来越多地用于生物医学研究，并提供了优势，因为它们 然而，尽管有这些优势，啮齿动物模型的解剖学和生理学与人类非常相似。 主导了许多研究领域，包括神经科学，这可能部分归因于更大的影响。 在当前的应用中，研究人员可以使用许多可供啮齿动物研究人员使用的技术。 提议创建一项新技术来促进大型动物研究人员并弥合这一差距。 具体来说，她建议培育具有红移通道视紫红质-黄水晶融合的转基因猪 蛋白质或绿色荧光蛋白/钙调蛋白传感器在神经元中表达，这将允许。 用于同时可视化神经元（及其神经支配），以及精确控制或 红移通道视紫红质变体的一个重要优点是它 可以被近远红光（~630 nm）激活，从而减少光毒性事件并打开 此外，神经激活的微创方法（即通过皮肤）的大门。 绿色荧光蛋白衍生物，黄水晶，将允许识别特定的神经元件 表达通道视紫红质，并实现器官神经支配的可视化。 转基因猪，研究人员建议使用 PiggyBac 靶向猪胎儿成纤维细胞 转座子技术。piggyBac 转座子系统允许“剪切和粘贴”集成。 这种方法的一个优点是它促进了稳定。 转基因猪将源自目标胎儿成纤维细胞。 密苏里大学 Randall Prather 博士和他的团队将进行体细胞核实验 含有目标核材料的胚胎将被转移到代孕母猪中并进行移植。 然后将允许发育至足月的转基因猪进行研究和表征。 佛罗里达大学研究人员实验室的拟议工作完全符合。 SPARC 的优先事项，并将促进末梢器官的周围神经成像和靶向 此外，它的用途不仅限于周围神经系统研究人员， 由于中枢神经系统神经元也会表达视紫红质-黄水晶融合蛋白或绿色通道 因此，本申请中提出的工作具有荧光蛋白/钙调蛋白传感器。 具有极大加速神经科学领域发展并推动实验室到临床进程的潜力。