The objectives of this proposal are focussed on gene regulation in murine embryonal carcinoma (EC) cells as a model system using recombinant viral genes as molecular probes. EC cells are equivalent to the embryonic stem cells and viral genes are useful probes because undifferentiated EC cells cannot express the genes of certain viruses (e.g. retroviruses) but they can do so once differentiated. To understand the molecular mechanisms of the block to the viral gene expression, novel EC cell clones were isolated: By infection of EC cells with a recombinant transducing retrovirus that carries the neomycin resistance (neo) gene linked to the Moloney murine leukemia virus (M-MuLV) LTR, transductant EC clones that grew in the presence of a neomycin analogue, G418 were isolated. These transductant clonal lines are as undifferentiated as the parental EC cells according to the criteria tested. The expression of the neo gene of the recombinant provirus can be explained by cis-acting mechanisms. These provirus genomes have been molecularly cloned together with the flanking cellular sequences. Many of these lambda clones transfected parental EC cell lines at much higher efficiency than control plasmid with LTR-neo sequence. Two possible mechanisms are suggested: (a) the provirus genome is integrated in a vicinity of a cellular enhancer element, and/or (b) the provirus genome has obtained mutation/rearrangement in LTR regulatory region. To test these possibilities, the 5' flanking cellular sequence has been isolated from one of such clones and inserted into a test plasmid at a location upstream of LTR-neo sequence. With this construction, the transfection efficiency increased 50 to 100 fold, independent of the fragment orientation. Deletion analysis indicated that the enhancer activity was localized in a region of less than 1.5kb. Its sequence is currently being determined. Rearrangements of proviral genomes were also found in many of the transductant EC cell clones. More enhancer-like elements will be isolated from other clones, their structures determined, activity characterized, and the cellular genes under the control of these enhancer elements will be identified. The structure and biological activity of the rearranged proviral regulatory sequences will also be determined. These studies will give us basic insights how gene expression is controlled in mammalian embryonic stem cells and will provide us with a means to construct a new series of expression vectors useful for gene therapy.
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