Effects of homology length and donor vector arrangement on the efficiency of double-strand break-mediated recombination in human cells.
We use an Epstein-Barr virus (EBV) plasmid model chromosome system to study how different donor plasmid constructs affect recombination stimulated by an I-SceI-induced double-strand break in the target sequence in human cells. The entire 3.5 kb lacZ gene was efficiently recombined into a target EBV vector lacking lacZ sequences, but having limited homology to the donor plasmid. A donor plasmid with lacZ flanked by sequence homologous to the target consistently generated gene conversion events and was more effective than a donor carrying lacZ outside the same sequence homology. Reducing the length of homology between the target and donor from 5.5 kb to 1 kb caused only a 3-fold drop in recombination frequency, contrasting with the exponential dependence on homology length seen when no DSB is present in the target. These results document a DSB-induced 175-fold increase in recombination of a heterologous gene into a target, requiring only limited flanking homology.
Department of Genetics, Stanford University School of Medicine, Stanford, California 94305-5120, USA.
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JE
Phillips
M P
MP
Calos
D002478:Cells, Cultured; D002934:Cinnamates; D004249:DNA Damage; D004351:Drug Resistance; D019309:Epstein-Barr Virus Nuclear Antigens / Q000235:genetics; D005822:Genetic Vectors; D004854:Herpesvirus 4, Human / Q000235:genetics; D006801:Humans; D006921:Hygromycin B / Q000031:analogs & derivatives / Q000494:pharmacology; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D007612:Kanamycin / Q000494:pharmacology; D007763:Lac Operon; D010957:Plasmids / Q000235:genetics; D011995:Recombination, Genetic; D012689:Sequence Homology, Nucleic Acid
D002934:Cinnamates; D019309:Epstein-Barr Virus Nuclear Antigens; D006921:Hygromycin B; C026273:hygromycin A; D007612:Kanamycin; D007041:Hypoxanthine Phosphoribosyltransferase; C417029:EBV-encoded nuclear antigen 1