Sinclair, Angus McLeod; (1994) Cloning and the characterization of the regulatory elements of the Ly-6E.1 (Sca-1) gene. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Pluripotent haematopoietic stem cells (HSCs) differentiate into all mature blood cell lineages and have been characterized as the subpopulation of murine bone marrow cells expressing cell surface markers Sca-1+, Thy-1lo and Lin-. Stem cell antigen Sca-1, is one of the earliest differentiation markers detected on the surface of both fetal liver and adult bone marrow HSCs. Expression of this antigen also occurs on thymocyte progenitors, activated lymphocytes and on some non-haematopoietic tissues. The alloantigens detected by the Sca-1 monoclonal antibody are encoded by strain specific Ly-6E.1 and Ly-6A.2 alleles, members of the Ly-6 multigene family. The characterization and isolation of the regulatory elements of Ly-6E.1 and Ly-6A.2 that govern tissue specific and high level expression in the haematopoietic system, particularly stem cells, are therefore of considerable interest. In order to study the control elements of the Ly-6E.1 allele, a 30 kb fragment encoding a fully functional gene with 13 kb of 5' and 13 kb of 3' flanking sequence, has been cloned. Transfection analysis in a murine erythroleukaemic (MEL) cell line has demonstrated that a 14 kb BamHI fragment from this clone is sufficient to confer high levels of Ly-6E.1 gene expression on γ-interferon induction. To identify elements involved in Ly-6E.1 and Ly-6A.2 gene regulation, extensive DNase I hypersensitive site mapping of both expressing and non-expressing haematopoietic cell lines has been performed. This hypersensitive site analysis has demonstrated that 5' and 3' sites correlate with antigen expression in FDCP-1 cells, MEL cells and various T cell lines. Furthermore, allelic differences in hypersensitivity occur within the 5' flanking chromatin of the Ly-6A.2 and Ly-6E.1 alleles after γ-interferon induction and suggests a mechanism for the regulation of differential antigen expression within the haematopoietic system. Reporter constructs containing various 5' and 3' regions of the Ly-6E.1 gene have been analysed in γ-interferon inducible MEL and constitutively expressing N1H3T3 cell lines. These studies have demonstrated that Ly-6E.1 downstream sites contain a γ-interferon inducible element which synergizes with the promoter GAS element to confer high level γ-interferon induced Ly-6E.1 expression in MEL cells. Furthermore, preliminary in vivo analysis has demonstrated that the 5' and 3' Ly-6E.1 hypersensitive sites contain the necessary elements to direct tissue specific expression to the mature adult lymphoid system, bone marrow and other tissues, in addition to early embryonic mesonephros (AGM) and fetal liver (Miles-personal communication). These data strongly suggest that the Ly-6E.1 regulatory elements identified and characterized within this thesis, can be used to direct heterologous gene expression to stem cells.

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