Epstein-Barr virus plasmid model system for analyzing recombination in human cells

Homologous recombination stimulated by a double-strand break at a desired t arget site offers a method to achieve site-specific integration useful for gene therapy and other genetic engineering. To test parameters needed for t his strategy, we developed an Epstein-Barr virus shuttle vector model syste m as a generic tool. This extrachromosomal plasmid assay system has several advantages over a chromosomal assay. The system detects all classes of rec ombination events without selection and allows rapid analysis of the freque ncy and nature of recombination events. We found that a double-strand break at the target site stimulated a large increase in recombination frequency. The resulting recombinants included one-sided insertion events, as well as two-sided or gene conversion events. A circular donor substrate was more e ffective in recombination than linearized donor DNA. (C) 1999 Academic Pres s.