This physician Scientist Award proposal consists of a two phase plan designed to prepare the candidate for a career as an independent investigator. Phase I of this plan would constitute a well balanced didactic training program in molecular biology combined with a period of intensive laboratory training in molecular genetics (specifically focusing on the CD18 family of leukocyte adhesion molecules) to give the candidate the experience and guidance required for initiation of independent research. Phase II of the program will involve further in-depth studies of the biology of the cell surface adhesion molecules investigated during Phase I, with an emphasis on exploring the role these substances play in inflammatory responses in the rheumatic disease. During Phase I the candidate will initiate studies of the molecular genetics of the disease leukocyte adhesion deficiency (LAD) in this laboratory. This disease is characterized by a defect in adhesion- related functions of lymphocytes, monocytes and granulocytes resulting in recurrent life-threatening bacterial infections in affected patients. A defect in the beta-subunit of certain cell surface adhesion proteins has been identified as responsible for the leukocyte dysfunction. The human gene for this protein has been cloned, and the cDNA is available in our laboratory.
The specific aims of this project will be: 1) Insertion of the intact cDNA into appropriate expression vectors followed by in vitro gene transfer of these constructs into cultured cells to document expression; 2) Transfection of the constructs into ecotropic packaging cell lines, producing retroviral vectors for use in bone marrow transplantation mediated gene transfer of the human beta-subunit protein into mice; 3) Cloning of the mouse gene for the homologue of this protein using the human cDNA as a probe; and 4) Using site-directed mutagenesis and germ line gene transfer to produce a mouse model of the gene defect. This mouse model most importantly would facilitate studies of the structure and function of the integrin family of cell surface adhesion molecules, and secondarily would provide a mouse model of LAD, permitting studies of gene therapy in the disease. The candidate will have gained considerable experience in molecular genetic research during the initial two years of work under Phase I. During Phase II, he will continue the work with the leukocyte adhesion molecules directing attention to completion of the development of the CD18-deficient mouse, and the application of this model and the acquired skills to study the mechanisms of inflammation.
|Wilson, R W; Ballantyne, C M; Smith, C W et al. (1993) Gene targeting yields a CD18-mutant mouse for study of inflammation. J Immunol 151:1571-8|
|Yorifuji, T; Wilson, R W; Beaudet, A L (1993) Retroviral mediated expression of CD18 in normal and deficient human bone marrow progenitor cells. Hum Mol Genet 2:1443-8|
|Sanders, W E; Wilson, R W; Ballantyne, C M et al. (1992) Molecular cloning and analysis of in vivo expression of murine P-selectin. Blood 80:795-800|
|Wilson, R W; O'Brien, W E; Beaudet, A L (1989) Nucleotide sequence of the cDNA from the mouse leukocyte adhesion protein CD18. Nucleic Acids Res 17:5397|