Platelets play a major role both in health and in disease, and their therapeutic use represents one of the most vexing problems in transfusion medicine today. Despite this, very little is known about the molecular mechanisms that govern megakaryocytopoiesis and platelet formation. This lack of understanding is largely due to the lack of systems in which to perform in vivo and in vitro analysis. We propose to develop and use transgenic mouse lines that will allow us to study the molecular regulation of megakaryocytopoiesis in vivo. Using promoters with transcriptional activity restricted to cells of the megakaryocyte lineage (platelet factor 4, GPIIb, GPIIa, GPIX, and Mpl) we will develop transgenic mice that express the subgroup A, avian leukosis virus receptor (ALV-R) restricted to the surface of cells in the megakaryocyte developmental pathway. The mice will be fully characterized for ALV-R expression, and conditions will be developed for optimal ALV infection. ALV vectors can then be used to introduce any gene of interest into the mice and have it expressed only in cells of the megakaryocyte lineage. We will use the mice for in vivo studies of: i) the mechanism of action of Mpl, the recently identified thrombopoietin receptor, and ii) the role of Mark/Hyl, a recently identified non-receptor tyrosine kinase, in megakaryocytopoiesis and platelet function. By interbreeding the ALV-R-expressing mice with mpl-/- mice and with matk/hyl-/- mice we will be able to introduce wild type and mutant alleles of mpl and matk/hyl into a null genetic background. This approach will allow us to perform in vivo structure/function studies of Mpl protein in the absence of a complicating wild type protein. A similar approach will also allow us to study signaling via Mpl and to determine the role of Matk/Hylin intracellular signaling during megakaryocyte development. The transgenic mice will be applicable to the study fee any gene thought to be involved in megakaryocytopoiesis, and they can also serve as a model for gene therapy to correct congenital platelet defects. We will also use the ALV-R transgenic mice, and other mice already available, to develop immortalized megakaryocytic cell liens for in vitro studies.
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