Protocol

Preparation of Recombinant Baculoviruses with the BVboost System

Adapted from Gene Transfer (eds. Friedmann and Rossi). CSHL Press, Cold Spring Harbor, NY, USA, 2007.

INTRODUCTION

We have developed an improved transposition-based system (BVboost) for the generation of recombinant baculoviruses. This system bypasses the disadvantages of the original transposition-based generation of baculoviral genomes in Escherichia coli while remaining a simple, rapid, and convenient viral production technique. The method is based on the modified donor vector (pBVboost) and an improved selection scheme of the baculoviral genomes (bacmids) in E. coli with a mutated sacB gene. Recombinant bacmids can be generated at a frequency of ~10⁷/µg of donor vector with a negligible background. The BVboost system also allows efficient setups for high-throughput screening and gene expression purposes. After cloning the desired gene/cDNA/library into a BVboost system-compatible donor plasmid, the recombinant baculoviral genome is prepared simply by transforming electrocompetent DH10BacTn7 E. coli cells with the donor. Transfer from the donor vector into the baculoviral genome (bacmid) occurs via a Tn7-mediated site-specific transposition reaction in E. coli cells. The selection scheme guarantees that virtually all colonies are correct. The recombinant baculoviral genome is subsequently extracted from E. coli culture using a modified isolation procedure for large plasmids. To generate the recombinant viruses, insect cells are transfected with the isolated recombinant bacmid. This protocol provides instructions on how to prepare recombinant baculoviruses by the BVboost system in order to express the desired gene(s) in insect and/or vertebrate cells.