TARGETED MUTATION OF TGF-BETA-1 IN ES CELLS

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.