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Epstein-Barr virus plasmid model system for analyzing recombination in human cells
J E Phillips1, B Thyagarajan, M P Calos
1Department of Genetics, Stanford University School of Medicine, Stanford, California 94305-5120, USA.
Plasmid
|June 15, 1999
Summary
Site-specific integration using homologous recombination is enhanced by double-strand breaks at target sites. An Epstein-Barr virus shuttle vector system efficiently analyzes recombination events, favoring circular donor DNA.
Area of Science:
- Molecular Biology
- Genetic Engineering
- Gene Therapy
Background:
- Homologous recombination enables site-specific DNA integration, crucial for gene therapy.
- Double-strand breaks at target sites can stimulate recombination efficiency.
Purpose of the Study:
- To develop and utilize an Epstein-Barr virus (EBV) shuttle vector system for studying site-specific integration.
- To investigate the parameters influencing homologous recombination frequency and outcomes.
Main Methods:
- Development of an extrachromosomal Epstein-Barr virus shuttle vector assay.
- Analysis of recombination events, including one-sided insertions, two-sided integrations, and gene conversion.
- Comparison of circular versus linearized donor DNA substrates for recombination efficiency.
Main Results:
- A double-strand break at the target site significantly increased recombination frequency.
- The EBV shuttle vector system detected all recombination classes without selection.
- Circular donor DNA was more effective for recombination than linearized DNA.
Conclusions:
- The EBV shuttle vector system is a powerful tool for analyzing site-specific recombination.
- Double-strand breaks are effective in stimulating homologous recombination for genetic engineering.
- Optimizing donor DNA structure, such as using circular forms, enhances recombination efficiency.