Cloning and functional analysis of the promoter region of an intestinal stem cell specific expressed gene,Musashi-1 / 肠外与肠内营养

ABSTRACT

Objective:To clone and functionally analyze the promoter region of an intestinal specific expressed gene,Musashi-1.Methods:The 5' flanking region of MSI-1 gene was cloned from C57BL/6J mouse genomic DNA using PCR-mediated recombination.Expression of MSI-1 mRNA was determined in Colon26 and B16 cell lines using Northern Blotting.Various 5′-deletion recombination plasmids were constructed and transfected transiently into the Colon26 cell line.Luciferase reporter assay was performed to determine the relative transcriptional activities of various 5′-deltion fragments.Results:MSI-1 mRNA was expressed in both Colon26 and B16 cell lines,but much higher in Colon26 cell line.The transcriptional activity of DNA fragment from 73bp downstream to 4 939bp upstream the(MSI-1) gene transcriptional start site was 29.9 fold of the pGL3-basic empty vector.Conclusion:(pSMI+73~)-4 939 has the transcriptional activity and can be regarded as the promoter of MSI-1 gene.A cis-acting element lies between 4 011bp to 4 939 bp upstream the transcriptional start site of(MSI-1) gene,which may be responsible for the tissue specific expression of MSI-1 gene.The cloning of MSI-1 gene promoter is a precondition for the isolation and purification of intestinal stem cells using this promoter.