The major focus of this proposal is the development of murine retroviruses as vectors for introducing cloned genes into mammalian cells and the intact animal. Our proposed studies can be classified into two broad areas: I. The generation and characterization of vector systems useful for studying the expression and regulation of cloned DNA sequences (particularly transcriptional control sequences) and II. Studies on the general characteristics of retroviral mediated transformation. The studies to develop vectors compatible with the transfer of whole genomic and transcriptional control sequences will include a detailed analysis of factors which determine the biogenesis and transmissibility of retrovirus genomes. In addition, the effects of introducing transcriptionally active sequences into a retrovirus transcriptional unit will be assessed. To study transcriptional control sequences in isolation from their corresponding genes, vectors deficient in viral transcriptional activity will be utilized. A specific motivation of these studies is the development of retrovirus vectors with an expanded or restricted host range as a consequence of transcriptional activity. Recombinants containing different transcriptional specificities will be used in studies of oncogene function in vivo and for obtaining the expression of genes in the early mouse embyo. The transcriptionally defective vectors will also be used to isolate and characterize cellular enhancer sequences. To extend the general utility of the retrovirus vectors, experiments are proposed to develop additional dominant selectable markers and to widen the viral host range. The whole battery of recombinants constructed for the purposes of studying transcriptional regulation will be used in gene transfer studies both in cultured cells and the intact animal (insertion into the germ line). A major goal of the studies is to determine how the chromosomal site of integration of the transferred sequences affects those sequences expression and regulation. A related goal is to determine how multiply linked transcriptional enhancer sequences interact in vivo. A final series of experiments outlined in Section II of the proposal addresses the question of the nature and frequency of reverse transcriptase induced mutation of retrovirus genomes. A retrovirus shuttle vector system which permits the rapid scoring of mutations in bacteria will be utilized for these studies.

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Whitehead Institute for Biomedical Research
United States
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