Transgenic Pigs with Red-Shifted Channelrhodopsin-Citrine Fusion Proteins

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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.