High-frequency intrachromosomal gene conversion induced by triplex-forming oligonucleotides microinjected into mouse cells
| Autor: | Zhongjun Luo, Peter M. Glazer, A. Fawad Faruqi, Margaret A. Macris |
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| Rok vydání: | 2000 |
| Předmět: |
Recombination
Genetic Multidisciplinary Base Sequence Microinjections Oligonucleotide Mutant Gene Conversion Oligonucleotides Wild type Transfection Biological Sciences Biology Thymidine Kinase Molecular biology Cell Line Mice Thymidine kinase Animals Nucleic Acid Conformation Simplexvirus Direct repeat Gene conversion Gene DNA Primers |
| Zdroj: | Proceedings of the National Academy of Sciences. 97:9003-9008 |
| ISSN: | 1091-6490 0027-8424 |
| DOI: | 10.1073/pnas.160004997 |
| Popis: | 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 × 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. |
| Databáze: | OpenAIRE |
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