Establishing the Neurogenesis Pathways in C17.2 Neural Stem Cell by Innovating a High Efficient Gene Entrapment System

Given that more than 10 billions of neurons constitute a human brain, it is a formidable task to study the establishment of human neural network for ultimately understanding the physiological process of learning, memory, and cognition. In the past few decades, studies using simple model animals, such as fruit flies and nematodes, have gained great triumphs in the field of neuroscience. Although the simplicity of neural structures and networks in those model animals makes it convenient to study their neural functions, findings observed in these simple animal models are often unable to be extrapolated to understand the exquisite and far more complicated neural networks of mammalian brains. Hence, it is a devastating need to establish systems dedicated to tackle the development of mammalian brains and to complement the limitations encountered by using oversimplified animal models. Utilizing animal models to study gene function heavily relies on the easily identified phenotypes. Unfortunately, a gene disruption in animal models, especially in a more complicated model, such as mice, often results in phenotypes that are easily missed and in turn fails to address potential gene functions. To overcome the insufficient information provided from animal models alone as well as to avoid directly studying the over complicated human neural networks, in this study I propose a feasible task: identifying key genes involving in terminal neural differentiation by developing and performing a genetic screen using a novel and highly efficient gene trap system that is dedicated to trap genes involving in a specific biological process or a particular differentiation pathway. Maintaining the pluripotency of a neuronal stem cell requires the highly differentiated neural genes to be silenced. Therefore, trapping genes involving in the terminal neuronal differentiating process relies on a system enabling targeting genes that are silenced in undifferentiated cells but are expressed in cells undergoing terminal neural differentiation. To achieve this goal, a drug-free piggyBac transposon-mediated genetic engineering system will be developed to target key genes involving in the terminal neural differentiation in doxycycline regulated REST-VP16 expressing C17.2 cells, a mouse neural stem-like cell line engineered to produce a recombinant REST-VP16 fusion protein that induce neural maturation. Using this novel gene-trapped host-vector platform, a library of trapped RESTVP16-engineered C17.2 clones will be established. To identify genes involving in the terminal neural differentiation, individual clones in the gene trap library will be induced to neurons in the absence of doxycycline but the presence of retinoic acid. As detailed in specific aims of this proposal, during the progression of terminal neurogenesis, the piggyBac reporter system trapped in genes involving in this process will be turned on and in turn facilitate the identification of desired clones for further isolation. Based on the timing of trapped gene expression during terminal neural differentiation, the pathway of the final stage of neurogenesis in C17.2 will be established. The success in this proposal not only will provide invaluable insights to the basic neurobiology but will also benefit the clinical treatments in the cell-based regenerative therapy. Additionally, this study will establish a paradigm that can be readily applied to identify key players involving in any given biological pathway.

Project ID:PA9804-0073
External Project ID:NSC98-2311-B182-001