The objective of the research project is to study the pathogenesis of severe combined immunodeficiency disease that is associated with adenosine deaminase deficiency. We propose to develop a mouse model for adenosine deaminase deficiency by the techniques of homologous recombination and chimera production via blastocyst injection of cultured mouse embryonic stem cells. Several mutant subclones of mouse embryonic stem cells have been derived. These clones will be characterized by adenosine deaminase assays, polymerase chain reaction and Southern hybridization. The clones that contain insertional mutation or point mutation in one of the two adenosine deaminase genes will be used to generate chimeric mice by micro- injection into blastocysts of a different genetic background, and reim- plantation of the embryo into a pseudopregnant mother. The chimeric mice will be bred to derive homozygous mutants lacking adenosine deaminase. The mouse model will be important to prove the causal relation between the enzyme deficiency and immunodeficiency. It will be useful to test dif- ferent hypotheses explaining the molecular pathology of the immunodefi- ciency disease. One such hypothesis to be tested in this proposal is that deoxyadenosine causes immunodeficiency by interrupting the signal trans- duction mechanism in lymphocytes. Finally, the animal model will be valuable in the development of gene therapy of ADA deficiency in humans.
