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

Abstract

Homologous recombination stimulated by a double-strand break at a desired target site offers a method to achieve site-specific integration useful for gene therapy and other genetic engineering. To test parameters needed for this strategy, we developed an Epstein-Barr virus shuttle vector model system as a genetic tool. This extrachromosomal plasmid assay system has several advantages over a chromosomal assay. The system detects all classes of recombination events without selection and allows rapid analysis of the frequency 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 effective in recombination than linearized donor DNA.