project 1

1. Reverse Genetic Screening to Determine the Vascular Functions of Novel Flow-Sensitive Genes in Zebrafish

The mechanical forces have been thought to be an intrigue environmental stimulation which controls physiological functions of endothelial cells. Among which, laminar shear stress generated by blood flow is critical for normal endothelium function. Indeed, it has been shown that the formation of pathological atherosclerotic plaque is associated with disturbed flow pattern, which suggests this vascular disease is resulted from the aberrant response of endothelial cells to different mechanical forces. However, the detailed mechanisms have not been fully elucidated given many flow-dependent genes and pathways have been reported but still a huge set of similar genes have not been studied at all. One major limitation to this is the lack of a simple model for in vivo phenotypical analysis. In addition, using mouse for specific gene knock-out study requires a significant time and efforts which prevent a strategy for gene screening. Fortunately, recent studies using the CRISPR/Cas9 system demonstrated an efficient method of targeted gene disruption. Further, zebrafish has characteristics that makes it an ideal model organism for studying genetic determinants that participates in development and disease. In this study, we are in the process to generate targeted mutations throughout zebrafish genome and perform a revise genetic screen on selected novel flow-dependent genes to determine their functions related to vascular diseases. A major goal of the proposed studies will be determining vascular phenotype of selected flow-dependent genes associated to any human vascular disease in order to create a platform for future drug screening. We are now streamlining the process of making gene knockouts and validating the generated mutants as null alleles by examining their expression level. We will also identify the potential off-target sites through the zebrafish genome sequence and evaluate the frequency of off-target effect. We will further perform a comprehensive phenotypic analysis in vascular system and determine the autonomous role of endothelial cells for these novel flow-dependent genes. By creating the proposed mutant lines, this study will reveal new functions of selected novel flow-dependent genes and ultimately can be used to create new model platforms for future drug screening on the mutants with vascular defects.  This study will have far-reaching significance in vascular diseases related to mechanobiology.