G. Wang et al., TARGETED MUTAGENESIS IN MAMMALIAN-CELLS MEDIATED BY INTRACELLULAR TRIPLE-HELIX FORMATION, Molecular and cellular biology, 15(3), 1995, pp. 1759-1768
As an alternative to standard gene transfer techniques for genetic man
ipulation, we have investigated the use of triple helix forming oligon
ucleotides to target mutations to selected genes within mammalian cell
s. By treating monkey COS cells with oligonucleotides linked to psoral
en, we have generated targeted mutations in a simian virus 40 (SV40) v
ector contained within the cells via intracellular triple helix format
ion. Oligonucleotide entry into the cells and sequence-specific triple
r formation within the SV40 DNA deliver the psoralen to the targeted s
ite. Photoactivation of the psoralen by long wavelength UV light yield
s adducts and thereby mutations at that site. We engineered into the S
V40 vector novel supF mutation reporter genes containing modified poly
purine sites amenable to tripler formation. By comparing the abilities
of a series of oligonucleotides to target these new sites, we show th
at targeted mutagenesis in vivo depends on the strength and specificit
y of the third strand binding. Oligonucleotides with weak target site
binding affinity or with only partial target site homology were ineffe
ctive at inducing mutations in the SV40 vectors within the COS cells.
We also show that the targeted mutagenesis is dependent on the oligonu
cleotide concentration and is influenced by the timing of the oligonuc
leotide treatment and of the UV irradiation of the cells. Frequencies
of intracellular targeted mutagenesis in the range of 1 to 2% were obs
erved, depending upon the conditions of the experiment. DNA sequence a
nalysis revealed that most of the mutations were T.A-to-A.T transversi
ons precisely at the targeted psoralen intercalation site. Several del
etions encompassing that site were also seen. The ability to target mu
tations to selected sites within mammalian cells by using modified tri
pler-forming oligonucleotides may provide a new research tool and may
eventually lead to therapeutic applications.