The production of mature blood cells depends on the activation of a developmental hierarchy of precursor cells. The cells at the root of this hierarchy - hematopoietic stem cells - proliferate and differentiate to form progenitors with progressively restricted developmental potential. The identification of stem cell-specific growth regulators, especially those influencing self-renewal replication rather than differentiation, is an important practical and theoretical problem. Genes expressed differentially between closely related cell types often are important in the functional differences between them. These genes may themselves be """"""""master"""""""" genes or be directly regulated by coordinating genetic programs. This proposal describes a novel system to simultaneously isolate and mutate genes expressed selectively in hematopoietic precursors or in cells inducing those precursors. Gene trap retrovirus vectors carrying a Herpes simplex virus thymidine kinase/beta-galactosidase fusion gene (galtek) will be used to transduce undifferentiated murine embryonic stem (ES) cells. Individual ES cell clones will be cultured under conditions that are permissive for the formation of hematopoietically-active embryoid bodies (EB). Approximately 10,000 transduced ES clones will be screened by examining the phenotype of embryoid bodies with or without addition of FIAU, a drug that is selectively toxic to cells expressing thymidine kinase. Under selection in FIAU, clones with integrations allowing expression of galtek in hematopoietic precursors will form EB with suppressed hematopoiesis. The pattern of expression of the targeted genes will be studied using in situ hybridization and histochemical staining for vector-encoded beta-galactosidase. Specific tests for integration into a gene that is expressed in multilineage hematopoietic precursors will be carried out. In addition, the integration of a gene trap vector is usually mutagenic so that the ES cell clones will be used to test the function of the targeted genes in families of animals in which the gene trap insertion has been passed into the germline. ES clones with the appropriate differentiation phenotype will be used to isolate fusion transcripts providing specific probes for the cloning and sequencing of the endogenous target genes. We have established the general techniques for culture of the ES cells and the plausibility of the screening method. In approximately 850 clones screened to date, 4 have been found to have absent or delayed hematopoiesis in the presence of FIAU. Finally, as an alternative approach we will also use differential display PCR to identify cDNA clones that are uniquely expressed within a pool of highly purified murine hematopoietic precursors. Targeted genes and differentially expressed genes will be characterized by sequencing, determination of chromosomal position, analysis of upstream regulatory signals, and pattern of gene expression during development.
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