A major impediment to the study of mammalian embryogenesis has been the lack of developmental mutations that can be molecularly defined, and thus associated with a cloned gene and a protein product. In this proposal we describe procedures for the generation and analysis of developmental mutations that can be studied at both the molecular and cellular levels. Such mutations are produced by the introduction of a foreign DNA into the mouse germ line by the microinjection of mouse egg pronuclei. These mutations are likely to result from the inactivation of developmentally essential genes by the integration of the foreign DNA. A unique advantage of this technique is that the inactivated host gene may be cloned using the foreign DNA as a molecular probe. We have identified one transgenic mouse line that carries a recessive prenatal lethal mutation that was caused by the integration of a bacteriophage lambda recombinant, LambdaRBetaG2, containing the rabbit adult Beta-globin gene. The site in the mouse genome into which this foreign DNA has inserted is referred to as the RBeta3 locus. Animals homozygous for the foreign DNA insertion at the RBeta3 locus begin to arrest in development around the time of implantation and have died and been resorbed by the 8th day of gestation. We propose to use the LambdaRBetaG2 sequences as hybridization probes to molecularly clone the developmentally essential gene at the RBeta3 locus that has been inactivated by the integration of the foreign DNA. The mRNA and protein products of this gene will be characterized to determine the amino acid sequence of the protein as well as its tissue distribution and cellular location in the developing mouse embryo. This information will provide a framework for further studies on the function of this gene during mouse embryogenesis. We also propose to identify additional insertional mutations that affect early development of the mouse by generating new transgenic mouse lines. For these studies we will microinject a construct that will facilitate the molecular cloning and analysis of genes inactivated by the insertion of a foreign DNA. These studies promise to provide powerful new probes for the study of mammalian development at both the molecular and cellular levels.

National Institute of Health (NIH)
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
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Human Embryology and Development Subcommittee 2 (HED)
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Sloan-Kettering Institute for Cancer Research
New York
United States
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