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Journal Article | Research Support, Non-U.S. Gov't | Research Support, U.S. Gov't, P.H.S.

Targeted mutagenesis in mammalian cells mediated by intracellular triple helix formation.

G Wang, D D Levy, M M Seidman, P M Glazer
G Wang
Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, Connecticut 06510.
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D D Levy
Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, Connecticut 06510.
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M M Seidman
Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, Connecticut 06510.
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P M Glazer
Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, Connecticut 06510.
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DOI: 10.1128/MCB.15.3.1759
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ABSTRACT

As an alternative to standard gene transfer techniques for genetic manipulation, we have investigated the use of triple helix-forming oligonucleotides to target mutations to selected genes within mammalian cells. By treating monkey COS cells with oligonucleotides linked to psoralen, we have generated targeted mutations in a simian virus 40 (SV40) vector contained within the cells via intracellular triple helix formation. Oligonucleotide entry into the cells and sequence-specific triplex formation within the SV40 DNA deliver the psoralen to the targeted site. Photoactivation of the psoralen by long-wavelength UV light yields adducts and thereby mutations at that site. We engineered into the SV40 vector novel supF mutation reporter genes containing modified polypurine sites amenable to triplex formation. By comparing the abilities of a series of oligonucleotides to target these new sites, we show that targeted mutagenesis in vivo depends on the strength and specificity of the third-strand binding. Oligonucleotides with weak target site binding affinity or with only partial target site homology were ineffective at inducing mutations in the SV40 vectors within the COS cells. We also show that the targeted mutagenesis is dependent on the oligonucleotide concentration and is influenced by the timing of the oligonucleotide treatment and of the UV irradiation of the cells. Frequencies of intracellular targeted mutagenesis in the range of 1 to 2% were observed, depending upon the conditions of the experiment. DNA sequence analysis revealed that most of the mutations were T.A-to-A.T transversions precisely at the targeted psoralen intercalation site. Several deletions encompassing that site were also seen. The ability to target mutations to selected sites within mammalian cells by using modified triplex-forming oligonucleotides may provide a new research tool and may eventually lead to therapeutic applications.

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Targeted mutagenesis in mammalian cells mediated by intracellular triple helix formation.
G Wang, D D Levy, M M Seidman, P M Glazer
Molecular and Cellular Biology Mar 1995, 15 (3) 1759-1768; DOI: 10.1128/MCB.15.3.1759

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Targeted mutagenesis in mammalian cells mediated by intracellular triple helix formation.
G Wang, D D Levy, M M Seidman, P M Glazer
Molecular and Cellular Biology Mar 1995, 15 (3) 1759-1768; DOI: 10.1128/MCB.15.3.1759
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