Identifying Biomarkers/Genes Involving in Brain Formation and Function via a Piggybac-Based in Vivo Genetic Screen in Living Mice

During brain development, by responding to the molecular cues exist in the surrounding milieus the intrinsic genetic program of each neural cell triggers a series of gene regulation cascade that specifies their cell fate, instructs their further differentiation, and guide them to their final destination where they form synapses with their correct partners. As a consequence, an error-free neural network is established and a functional brain is formed. In the past two decades, most studies on brain development mainly relied on the use of simple model animals, such as Drosophila or C. elegans, to tackle these issues. Fruitful findings obtained from these studies have laid the foundation of current knowledge in neuroscience. The nervous system of these simple model animal is however far too simple to fully represent the complexity of a human brain. Given the availability of genetics and the structural and functional similarity of its nervous system as compared to a human brain, a mouse model is widely used to unfold the mystery of the formation and function of a human brain. Such studies rely on the phenotypic analysis of knockout mice generated either by homologous targeting or gene trap to uncover the genes involving in brain development and function. These types of studies involve costly, time consuming, and tedious processes that only can generate handful knockout mouse lines at a time. Once generated, these mutant mouse lines need to be elaborately analyzed one by one to determine whether there is any defect in the brain development of the these mutant mice. Unfolding the mystery of the function and development of a human brain requires the identification of all of the players involving in these biological processes. Hence, an innovated genetic screen in an in vivo setting is urgently needed to facility the search for regulatory genes as well as the biomarkers during different developmental stages. Accordingly, we propose to develop an in vivo gene entrapment system for establishing an immortalized neuronal gene trap library from mouse brains. This study will likely facilitate the identification of the neural biomarkers involving in brain development and enrich our understanding regarding the property of neural stem cell and the neural differentiation process. In this study, the piggyBac transposon will be adapted to achieve this goal by developing a highly efficient in vivo gene trap system in living mouse brains. With the expression of luciferase gene under the control of trapped promoter and constitutive expression of c-myc gene, the trapped neural cells will be able to form the luciferase positive colonies in mouse brains receiving the in vivo gene trap system. The brains with luciferase positive colonies will be detected lively by Magnetic resonance imaging (MRI) and be sacrificed to isolate luciferase positive clones. Thus, the genes trapped in the in vivo gene trap neural cell library will represent the set of genes expressed during brain development. In this proposal, we coordinate our expertise on piggyBac transposon system as well as the brain injection techniques to innovate a novel in vivo gene trap platform and conducting a high throughput genetic screen for key players involving in neuronal development in living mouse brains. This study will also allow us to reveal the in vivo gene expression profiles in a defined area of brains, hippocampus for instance, during brain development. Additionally, this study will likely facilitate the establishment of neural stem cell lines, and identify the biomarkers of various neural cell types at different developmental stages during brain formation. Furthermore, the success in obtaining those in vivo neural gene expression profiles as well as the biomarkers shall shed light on the development of therapeutic strategies for treating neural degenerative diseases.

Project ID:PC10101-1442
External Project ID:NSC99-2320-B182-010-MY3