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

为了分析m Kirre在体内的功能，我们着手破坏小鼠基因组中的m Kirre。我们筛选BAC文库，获得含有m Kirre基因的基因组片段，并分离出多个基因组片段。对m Kirre基因组片段进行限制性内切酶图谱分析，结合计算机数据库检索，完成了m Kirre基因座的基因组图谱。设计靶向载体以用新霉素盒替换第一外显子和5-启动子区的大部分以破坏m Kirre基因。将完成的靶向载体电穿孔到E14胚胎干（ES）细胞中，并且选择约400个新霉素抗性克隆并进行处理以通过Southern杂交鉴定同源重组克隆。我们获得了一个正确的靶向克隆，并通过与5 '-和3-探针以及Neo-探针杂交来证实。我们目前正在通过将该克隆注射到C57 BL/6小鼠的囊胚中来制备嵌合小鼠。比较表达ISF、ShIF或模拟质粒的MS 10细胞的表达谱，发现TIMP-3和SFRP-1被ISF过表达下调。通过在MS 10/ISF细胞中重新表达TIMP-3或SFRP-1基因，MS 10/ISF细胞的增强的造血干细胞（HSC）支持活性降低到与亲本MS 10细胞相似的水平。这些结果表明TIMP-3和SFRP-1在ISF赋予的增强的HSC活性中起关键作用。

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.