We have found in the past 4 years several genes that affect stem cell behaviors: bcl-2 inhibits programmed cell death, and increases HSC numbers and competitiveness in vivo; Wnt 3A and beta-catenin stimulate phenotypic HSC expansion in vitro, and these cells give LTMR upon transplantation; axin (an inhibitor of beta-catenin) blocks HSC proliferation in response to a variety of cytokines; bmi-1 (from Mike Clarke), whose absence prevents fetal liver hematopoiesis spreading to prenatal and postnatal bone marrow; and hTERT, which enforces HSC telomere maintenance through serial HSC transplants. We have begun the analysis of a number of genes with relative or absolute increased expression in LT-HSC vs. progeny (including ST-HSC). Here we plan to test whether our Wnt/beta -catenin/axin results define a manipulatable pathway for HSC self-renewal, and whether other identified (or newly identified) LT-HSC genes or leukemia translocated genes play roles in HSC self-renewal or other HSC behaviors. We plan to expand dramatically our efforts to find, catalogue, and determine cell lineage expression of all LT-HSC (and ST-HSC/MPP and CLP and CMP) transcripts. Finally, using tet-regulatable lentivirus vectors, we will test candidate genes by transfection of HSC, retain aliquots for in vivo hematopoiesis analyses, and co-transplant the rest to allow the whole animal select the most competitive clones. Recloning and retransfection will further limit the genes likely to play key roles in self-renewal. Recloning and retransfection will further limit the genes likely to play key roles in self-renewal, etc. ES cell complete or conditional knock-outs will help define the role of the genes in vivo.