Abstract
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.
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Phillips, J.E., Calos, M.P. Effects of Homology Length and Donor Vector Arrangement on the Efficiency of Double-Strand Break-Mediated Recombination in Human Cells. Somat Cell Mol Genet 25, 91–100 (1999). https://doi.org/10.1023/B:SCAM.0000007144.05961.2e
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DOI: https://doi.org/10.1023/B:SCAM.0000007144.05961.2e