Transgenic Pigs with Red-Shifted Channelrhodopsin-Citrine Fusion Proteins

项目摘要 大型动物越来越多地用于生物医学研究，并提供了一个优势， 解剖学和生理学与人类非常相似。然而，尽管有这些优势， 在包括神经科学在内的许多研究领域占据主导地位。这可能是因为， 啮齿动物研究人员可用的技术数量。在本申请中，研究人员 提出了一项新技术，以促进大型动物研究人员和弥合这一差距。 具体来说，她建议创造一种具有红移通道视紫红质-柠檬黄融合基因的转基因猪 蛋白质或神经元中表达的绿色荧光蛋白/钙调素蛋白传感器。这将允许 用于神经元（及其神经支配）的同时可视化，以及精确控制或 监控神经活动红移通道视紫红质变体的一个重要优点是， 可以被近远红光（~630 nm）激活，从而减少光毒性事件并打开 这为神经激活的微创方法（即通过皮肤）打开了大门。此外， 一种绿色荧光蛋白衍生物--柠檬黄，将允许识别特定的神经元件 表达通道视紫红质，以及使器官神经支配可视化。创建 转基因猪，研究者建议使用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”整合piggyBac转座子。 靶向构建体进入猪基因组。这种方法的一个优点是， 转基因表达转基因猪将来源于靶向的胎儿成纤维细胞， 密苏里州的大学，兰德尔·普拉瑟博士和他的团队将在那里进行体细胞核试验。 转移将含有目标核物质的胚胎转移到代孕母猪中， 允许其发展至足月。转基因猪随后将在 佛罗里达大学的研究者实验室。拟议的工作完全符合 优先考虑神经外科手术，并将有助于成像和靶向周围神经与终末器官 在大型动物模型中。此外，它的效用不仅限于周围神经系统研究人员， 因为中枢神经系统神经元也将表达通道视紫红质-柠檬酸融合蛋白或绿色 荧光蛋白/钙调素蛋白传感器。因此，本申请中提出的工作具有 有可能极大地加速神经科学领域的发展，并推动从实验室到临床的进程。

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.