Project Summary: Mouse genetic tools based on Cre recombinase and Cas9 nuclease have been applied for cell/tissue-specific gene knockout and cell lineage tracing. These tools enable control of gene expression and genome engineering to uncover detailed biology in a number of systems. However, the lack of precise spatiotemporal control hinders the broader applications of these tools. In this proposal, we will develop a new set of mouse genetic tools to address this issue. Then we propose to apply this system to investigate the establishment of the adult bone marrow hematopoietic stem cell (HSC) pool in vivo. Because HSCs self-renew and differentiate into all blood cell lineages throughout life, they are the basis for life-saving bone marrow transplantation in clinics. Therefore, understanding HSC biology has important translational implications. The establishment of the adult bone marrow HSC pool is essential for life-long hematopoiesis. However, how the adult bone marrow HSC pool is established has not been addressed. The lack of appropriate genetic tools to study HSCs at the clonal level in vivo has been a major hurdle. Our novel genetic tools will allow tracing HSCs at the clonal level in vivo. If successful, the results will significantly advance the field by directly addressing how the adult bone marrow HSC pool is established. Also, our proposed novel technologies will be widely applicable to various research fields beyond hematopoiesis to address fundamental questions.