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Site-specific insertion of DNA into a pseudorabies virus vector
1Central Research and Development Department, E. I. du Pont de Nemours and Company, Wilmington, DE 19898.
Summary
Researchers developed a novel method using Cre-lox recombination to insert and retrieve recombinant DNA in herpesvirus vectors. This technique enables efficient gene transfer and recovery in pseudorabies virus (PRV) for genetic engineering applications.
Area of Science:
- Molecular Biology
- Virology
- Genetic Engineering
Background:
- Herpesvirus vectors are valuable tools in molecular biology and gene therapy.
- Efficient methods for introducing and retrieving foreign DNA into these vectors are crucial for their application.
- The Cre-lox site-specific recombination system offers a precise mechanism for DNA manipulation.
Purpose of the Study:
- To develop a simple and efficient method for introducing recombinant DNA into a herpesvirus vector.
- To enable the retrieval of inserted DNA from the vector at a later time.
- To create a versatile tool for genetic manipulation of pseudorabies virus (PRV).
Main Methods:
- Utilized the Cre-lox site-specific recombination system of coliphage P1 for DNA insertion.
- Constructed a pseudorabies virus (PRV) vector (PRV42) with a lox site within the nonessential gIII gene.
- Incubated PRV42 DNA with Cre protein and a lox-containing plasmid for in vitro integration.
- Recovered infectious recombinant virus via transfection and identified it using a gIII-specific monoclonal antibody-based black-plaque assay.
Main Results:
- Achieved approximately 5% integration of plasmid DNA into the PRV genome at the lox site.
- Developed a nondestructive black-plaque assay for identifying recombinant viruses based on gIII epitope disruption.
- Demonstrated that the recombinant virus is infectious, stable, and exhibits growth comparable to the parental vector.
- Showcased efficient excision (greater than 50%) of inserted plasmid DNA from viral DNA using Cre for recovery.
Conclusions:
- The Cre-lox system provides an efficient method for in vitro DNA insertion into herpesvirus vectors.
- The developed method allows for the recovery and identification of infectious recombinant viruses.
- This technique offers a stable and effective platform for genetic engineering using pseudorabies virus.