Targeted gene conversion induced by triplex-directed psoralen interstrand crosslinks in mammalian cells.
Correction of a defective gene is a promising approach for both basic research and clinical gene therapy. However, the absence of site-specific targeting and the low efficiency of homologous recombination in human cells present barriers to successful gene targeting. In an effort to overcome these barriers, we utilized triplex-forming oligonucleotides (TFOs) conjugated to a DNA interstrand crosslinking (ICL) agent, psoralen (pTFO-ICLs), to improve the gene targeting efficiency at a specific site in DNA. Gene targeting events were monitored by the correction of a deletion on a recipient plasmid with the homologous sequence from a donor plasmid in human cells. The mechanism underlying this event is stimulation of homologous recombination by the pTFO-ICL. We found that pTFO-ICLs are efficient in inducing targeted gene conversion (GC) events in human cells. The deletion size in the recipient plasmid influenced both the recombination frequency and spectrum of recombinants; i.e. plasmids with smaller deletions had a higher frequency and proportion of GC events. The polarity of the pTFO-ICL also had a prominent effect on recombination. Our results suggest that pTFO-ICL induced intermolecular recombination provides an efficient method for targeted gene correction in mammalian cells.
Liu
Yaobin
Y
Department of Carcinogenesis, University of Texas MD Anderson Cancer Center, Science Park-Research Division, Smithville, Texas, USA.
Nairn
Rodney S
RS
Vasquez
Karen M
KM
D003432:Cross-Linking Reagents / Q000633:toxicity*; D004247:DNA / Q000378:metabolism; D005363:Ficusin / Q000633:toxicity*; D005785:Gene Conversion; D017353:Gene Deletion; D006367:HeLa Cells; D006801:Humans; D009841:Oligonucleotides / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic
D003432:Cross-Linking Reagents; D009841:Oligonucleotides; C051802:triplex DNA; D004247:DNA; D005363:Ficusin