High-frequency intrachromosomal gene conversion induced by triplex-forming oligonucleotides microinjected into mouse cells.
To test the ability of triple helix-forming oligonucleotides (TFOs) to promote recombination within chromosomal sites in mammalian cells, a mouse LTK(-) cell line was established carrying two mutant copies of the herpes simplex virus thymidine kinase (TK) gene as direct repeats in a single chromosomal locus. Recombination between these repeats can produce a functional TK gene and occurs at a spontaneous frequency of 4 x 10(-6) under standard culture conditions. When cells were microinjected with TFOs designed to bind to a 30-bp polypurine site situated between the two TK genes, recombination was observed at frequencies in the range of 1%, 2,500-fold above the background. Recombination was induced efficiently by injection of both psoralen-conjugated TFOs (followed by long-wave UVA light; 1. 2%) and unconjugated TFOs alone (1.0%). Control oligomers of scrambled sequence but identical base composition were ineffective, and no TFO-induced recombination was seen in a control LTK(-) cell line carrying an otherwise identical dual TK gene construct lacking the 30-bp polypurine target site. TFOs transfected with cationic lipids also induced recombinants in a highly sequence-specific manner but were less effective, with induced recombination frequencies of 6- to 7-fold over background. Examination of the TFO-induced recombinants by genomic Southern blotting revealed gene conversion events in which both TK genes were retained, but either the upstream (57%) or the downstream gene (43%) was corrected to wild type. These results suggest that, with efficient intracellular delivery, TFOs may be effective tools to promote site-specific recombination and targeted modification of chromosomal loci.
Luo
Z
Z
Departments of Therapeutic Radiology and Genetics, Yale University School of Medicine, P.O. Box 208040, New Haven, CT 06520-8040, USA.
Macris
M A
MA
Faruqi
A F
AF
Glazer
P M
PM
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D005785:Gene Conversion; D051379:Mice; D008845:Microinjections; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000008:administration & dosage*; D011995:Recombination, Genetic; D018139:Simplexvirus / Q000201:enzymology; D013937:Thymidine Kinase / Q000235:genetics
D017931:DNA Primers; D009841:Oligonucleotides; D013937:Thymidine Kinase