Construction of a recombinant adenovirus for efficient delivery of the I-SceI yeast endonuclease to human cells and its application in the in vivo cleavage of chromosomes to expose new potential telomeres.
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.
Anglana
M
M
Department of Pathology and Molecular Medicine, McMaster University, 1200 Main Street West, Hamilton, Ontario L8N 3Z5, Canada.
Bacchetti
S
S
D000256:Adenoviridae / Q000235:genetics* / Q000502:physiology; D001665:Binding Sites; D002460:Cell Line; D002877:Chromosomes, Human / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000235:genetics / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D011401:Promoter Regions, Genetic / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D016615:Telomere / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D014161:Transduction, Genetic; D014779:Virus Replication
D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific