Construction of a recombinant adenovirus for efficient delivery of the I-Scel yeast endonuclease to human cells and its application in the in vivo cleavage of chromosomes to expose new potential telomeres
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We have constructed a replication-defective adenovirus vector encoding the yeast I- Sce I endonuclease under the control of the murine cytomegalovirus immediate-early gene promoter (AdM Sce I) for efficient delivery of this enzyme to mammalian cells. We present evidence of AdM Sce I-mediated I- Sce I protein expression and cleavage activity in replication-permissive 293 cells, and of cleavage of chromosomes in vivo in both 293 cells and in non-permissive human cells. We have exploited this system for the generation of chromosomes capped by artificial telomeric sequences in cells with integrated plasmids containing telomeric DNA arrays adjacent to an I- Sce I recognition site. The properties of the AdM Sce I virus described here make it a useful tool for studying biological processes involving induction of DNA breaks, recombination and gene targeting in cells grown in culture and in vivo.
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