This project is directed toward the study of insertional mutagenesis in transgenic mice produced by pronuclear microinjection. Four projects are proposed. We have identified a transgenic pedigree with a recessive insertional mutation causing a motor abnormality and reduced male fertility. Genetic analysis demonstrates the mutation to be allelic with the spontaneous mouse mutation hotfoot (ho). Mouse DNA flanking the foreign gene insert has already been cloned. The first component of this proposal is to use the cloned DNA to isolate coding regions of the ho gene and determine the structure of the encoded protein as well as its developmental regulation and tissue distribution of expression. The second part of the application proposes a similar analysis of another insertional mutation in our transgenic colony which is allelic with Purkinje cell degeneration (pcd), a recessive disorder causing degeneration of cerebellar Purkinje cells, olfactory bulb mitral cells, and retinal photoreceptors. As with ho, flanking material has been cloned, and will be used to recover the coding sequence of the pcd gene. We also propose to develop a system for rapidly cloning material flanking genes introduced by microinjection. Such cloning is currently inefficient because when microinjected, DNA often integrates as very large concatamers. The strategy involves insertion of a selectable marker, the supF gene, into the mouse, as part of a vector designed so that the only fragment of the foreign gene which can be inserted into a bacteriophage cloning vehicle is that which is immediately linked to the host genomic material. A fourth series of experiments involves analysis of mouse DNA flanking transgenes. Similar studies of DNA adjacent to retrovirus proviral DNA has shown the material to be hypomethlyated, and to consist of sequences which are highly preferred for integration. We will analyze the mouse DNA adjoining microinjected transgenes to determine if it is hypomethylated, and/or if it contains sequences preferentially targeted for integration. This will determine if microinjection and retroviral infection are likely to produce the same pattern of insertional mutations. In addition, if sequences are identified which are especially prone to integration, they may be introduced into the germ line to serve as sites for directed integration, they may be introduced into the germ line to serve as sties for directed integration of genes in subsequent rounds of gene transfer.
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