The recombinational status of the genome of mammalian somatic tissues is thought to be static. The well studied exception is the site specific recombinations used to generate lymphoid antigen receptors. We have observed that a fully recombined immunoglobulin transgene is stable in its transmission in the germline. That is, the multiply integrated copies are consistently found in the kidney and tail DNA of over 8000 progeny. Thus, in the germline, the frequency of recombination within this tandem array resulting in a change of copy number is less than 10(- 3). However, we have discovered loss of transgene copies in certain somatic tissues of these mice at a very high frequency. The frequency with which losses occur approaches 0.5 in B lymphocytes. The work proposed here will yield insight into the factors important for genetic stability. We will utilize transgenes as reporter genes for studies of which specific cells and tissues of mice show instability. These studies will reveal the tissue type and/or expression requirements governing loss of transgene copies. We will investigate the tissue specificity of transgene loss by looking for such loss in clonal cell populations derived from various tissues at different stages of development. The findings will be extended by analysis of other transgenic mouse systems carrying transgenes whose characteristics overlap in defined ways with those of the transgene mentioned above. These studies will enable us to design modifications of our transgene to establish conclusively the sequence requirements for instability in this transgene. We will also attempt to analyze the possible mechanism of transgene deletion by investigating strand break sites and resolution products. Defining rules for transgene deletion, apart from its intrinsic interest in the analysis of the maintenance of genetic stability, is also of importance for transgene therapy. If potentially therapeutic transgenes delete at high frequency, the intended therapy may not be efficient. Currently, B lymphocytes are thought to be excellent expression systems for transgenes coding for soluble therapeutic products such as soluble complement or clotting factor components, soluble T cell receptor products or immunoglobulin. Thus, B cells are potential candidates for transgene therapy for a variety of deficiency diseases and autoimmune diseases. This project will define potential problems in their use in gene therapy in addition to establishing the mechanistic requirements for somatic transgene loss.
