Nitric oxide (NO) is now recognized as a major signal transduction molecule in mammalian systems. It has very widespread usage including the brain, endothelium, and phagocytic cells where it has been proposed to play roles in the learning process, blood pressure regulation, and tumor and infectious agent killing respectively. There have been three genes identified that produce NO from arginine. The enzyme responsible is called nitric oxide synthase (NOS). We propose to develop murine lines that are deficient in each of the three NOS genes to aid in our understanding of the processes in which NO participates. This will be accomplished using homologous recombination in embryonic stem cells with the subsequent generation of chimeras capable of transmitting the mutated genes to their offspring. To accomplish this each of three genes for the NOS loci will be isolated from genomic libraries and sufficiently characterized to allow for the preparation of the necessary molecular constructs. After establishing the murine lines, heterozygous mice will be bred to produce homozygous animals. The resulting litters will be closely monitored with particular concern for the possibility of embryonic lethality that might result from the homozygous deficiency of one of the NOS genes. Surviving animals will be genotyped and monitored for growth rate and other possible abnormalities. All animals will be subjected to complete autopsy at the time of death to detect any pathological lesions or other gross abnormalities. Depending on the particular NOS gene being studied we will perform relaxation studies on aortic rings, neutrophil emigration studies, response to infectious agents, or response to tumor growth. In addition, we propose to develop a murine line that is incapable of generating arginine from citrulline in non-hepatic tissue. This will be accomplished using a murine line that is deficient in argininosuccinate synthetase that we have recently obtained using the homologous recombination strategy and introducing a transgene that allows expression of AS in liver only. Finally, extensive breeding studies will be performed using the developed NOS lines and available murine lines deficient for such genes as the CD18, ICAM, p53, and retinoblastoma deficient lines and the ApoE deficient line. Such studies will provide extensive data concerning the role of NO in the physiology of the mammalian animal.
