Expression of the Ly-6e.1 (Sca-1) gene in transgenic mice for the study of haematopoietic stem cells during development

The haematopoietic stem cell is the progenitor of all mature blood cells. The processes involved in the maintenance and differentiation of haematopoietic stem cells throughout adult life are poorly understood and a greater understanding of these processes would be of clinical benefit for many haematological disorders. The Sca-1 antigen is a marker of murine haematopoietic stem cells and has been used in haematopoietic stem cell enrichment procedures. This thesis describes the production and analysis of transgenic mice containing DNA constructs based upon the Ly-6E.1 (Sca-1) gene. In the first series of experiments, the genetic regulatory elements of the Ly-6E.1 gene were studied by deletion analysis in vivo using Ly-6E.1/lacZ transgenes. The Ly-6E.1 transgene was shown to be functional in vivo and to possess a genetic element with some characteristics of a chromatin opening domain. More importantly, the Ly-6E.1 transgene was shown to be capable of directing heterologous gene expression to haematopoietic stem cells. Analysis of the expression pattern of Ly-6E.1/lacZ transgenes during development supports the notion of an intraembryonic origin for definitive haematopoiesis and suggests Ly-6E.1 may be a marker of haematopoietic stem cells throughout ontogeny. In the second series of experiments, transgenic mice were produced which expressed either the c-myc or the bcl-2 proto-oncogenes under the transcriptional control of the Ly-6E.l gene. All transgenic mice containing proto-oncogenes displayed perturbation of multiple haematopoietic lineages and the expression of these genes has facilitated the derivation of a novel cell line with a cell surface phenotype resembling that of a haematopoietic stem cell. These data demonstrate that the cloned 14Kb Ly-6E.1 gene is a useful tool for the study in vivo of the regulatory elements of a gene which is expressed in haematopoietic stem cells. Studies such as these could serve as a model leading to an understanding of the transcriptional programmes active in self-renewing, pluripotent haematopoietic stem cells. In addition, the results presented in this thesis provide evidence for the feasibility of using this genetic tool for the expression of heterologous genes in transgenic mice to study haematopoiesis in vivo and for the isolation of cell lines. Ultimately, such studies will enable the mechanisms of haematopoietic stem cell self-renewal and pluripotence to be understood