This research proposal involves the manipulation of the mammalian genome as a means to probe gene function. The overall aim of this project is to mutate by gene targeting the transforming growth factor-beta-1 (TGF-beta 1) gene in the mouse germline in order to understand the role that TGF-beta 1 plays in the control of growth, differentiation, and homeostasis in the embryo. The vehicle for accomplishing this is the mouse embryonic stem (ES) cell. The TGF-beta 1 gene will be altered in ES cells in a pre- determined manner by targeted gene mutation, and the mutated cells will be used to reconstitute an entire mouse, and to produce complex embryoid structures in culture. The developmental processes of growth and differentiation affected by this mutation will delineate the multiple functions of TGF-beta 1 in the complex settings of the cultured embryonic structures and of the transgenic embryos. (1) The first specific aim is to mutate by targeted gene modification one of the autosomal TGF-beta 1 genes in ES cells. (2) The second specific aim is to use the genetically modified ES cells to generate by blastocyst injection a transgenic mouse heterozygous for a functional TGF-beta 1 gene. (3) The third specific aim is to generate homozygous state in which the TGF-beta 1 expression is totally lacking. When this is done by breeding heterozygous animals to the homozygous state, the possible effects of TGF- beta 1 deficiency on pre-implantation embryos can be studied. When the second copy of the gene is mutated by a second round of gene targeting in ES cells, the possible effects of TGF-beta 1 deficiency on post- implantation embryogenesis can be investigated both in ES cells differentiating in culture, and in chimeric embryos produced by blastocyst injection. The homozygous state will also be generated in mosaic patches by two different approaches (i) within normal embryos by blastocyst injection of just one or two TGF-beta 1-deficient ES cells into normal blastocysts, thereby producing weakly chimeric animals; and (ii) in the offspring of irradiated pregnant females heterozygous for the TGF-beta 1 gene.
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