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Nucleic Acids Research Advance Access originally published online on September 2, 2009
Nucleic Acids Research 2009 37(19):6378-6388; doi:10.1093/nar/gkp678
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Nucleic Acids Research, 2009, Vol. 37, No. 19 6378-6388
© The Author(s) 2009. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Genome Integrity, Repair and Replication

Targeted gene conversion induced by triplex-directed psoralen interstrand crosslinks in mammalian cells

Yaobin Liu, Rodney S. Nairn and Karen M. Vasquez*

Department of Carcinogenesis, University of Texas M.D. Anderson Cancer Center, Science Park-Research Division, Smithville, Texas, USA

*To whom correspondence should be addressed. Tel: +1 512 237 9324; Fax: +1 512 237 2475; Email: kvasquez{at}mdanderson.org

Received June 19, 2009. Revised July 29, 2009. Accepted July 30, 2009.

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.


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