Regulation of self-renewal of hematopoietic stem cells by bone marrow stromal cells

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In order to analyze m Kirre function in vivo, we set out to disrupt m Kirre in mouse genome. We screened BAC library to obtain genomic fragment containing m Kirre gene and isolated several genomic fragments. Restriction mapping of m Kirre genomic fragment combined with the computer data base search completed the genomic map of m Kirre locus. Targeting vector was designed to replace the first exon and large portion of the 5-promoter region with Neomycin cassette to disrupt m Kirre gene. Completed targeting vector was electroporated into E14 embryonic stem (ES) cells, and approximately 400 Neomycin-resistant clones were selected and processed to identify homologously recombinant clones by Southern hybridization. We obtained one correctly targeted clone, which was confirmed by hybridizing with 5'- and 3-probes, together with Neo-probe. We are currently making chimeric mice by injecting this clone into blastcysts from C57BL/6 mice.As for ISF, we performed cDNA microarray analysis of bone marrow stromal cell lines overexpressing ISF and ShIF. Comparing expression profiles of MS10 cells expressing ISF, ShIF or mock plasmids revealed that TIMP-3 and SFRP-1 were down-regulated by ISF overexpression. By re-expressing TIMP-3 or SFRP-1 genes in MS10/ ISF cells, the enhanced hematopoietic stem cell (HSC) supporting activity of MS10/ ISF cells reduced a level similar to that of parental MS10 cells. These results indicate that TIMP-3 and SFRP-1 play critical roles in enhanced HSC activity conferred by ISF.