scientificprotocols authored about 8 years ago
Authors: Anna R. Mäkelä, Wolfgang Ernst, Reingard Grabherr and Christian Oker-Blom
Adapted from Gene Transfer: Delivery and Expression of DNA and RNA (ed. Friedmann and Rossi). CSHL Press, Cold Spring Harbor, NY, USA, 2007.
The baculovirus expression vector system has been used extensively to produce numerous proteins originating from both prokaryotic and eukaryotic sources. In addition to easy cloning techniques and abundant viral propagation, the system’s insect cell environment provides eukaryotic post-translational modification machinery. The baculovirus display vector system provides a number of advantages over prokaryotic systems, allowing the combination of genotype with phenotype, enabling presentation of foreign peptides or even complex proteins on the baculoviral envelope or capsid. Baculoviruses permit larger gene insertions, are easily propagated, and can be grown to high titers. Furthermore, surface modifications of the viral capsid enable specific targeting. This strategy can be used to enhance viral binding and entry to a wide variety of both dividing and nondividing mammalian cells as well as to produce antibodies against the displayed antigen. In addition, the technology should enable modifications of intracellular behavior, i.e., trafficking of recombinant “nanoparticles,” a highly relevant feature for studies of targeted gene or protein delivery. It is important to note that, although the viruses do not replicate in mammalian cells, they are not entirely transcriptionally silent. They can also be highly antigenic when used in vivo, limiting their therapeutic use. This protocol describes methods for generating display libraries.
An introduction to the theory and background of Baculovirus-Based Display and Gene Delivery Systems (Mäkelä et al. 2010a) is available. Additional procedures are presented for the Determination of Recombinant Baculovirus Display Viral Titer (Mäkelä et al. 2010b) and for the characterization of these gene delivery vehicles in Immunofluorescence Analysis of Baculovirus-Displayed Viral Proteins on Infected Insect Cells (Mäkelä et al. 2010c), Immunoelectron Microscopy Analysis of Recombinant Baculovirus Display Viruses (Mäkelä et al. 2010d), and Monitoring Baculovirus-Mediated Efficiency of Gene Delivery (Mäkelä et al. 2010e).
Figure 1. (A) Schematic of Ac-omega containing the Sce I single-cut site (sc) for direct gene insertion of DNA fragments treated with I-Sce I meganuclease. The polyhedrin promoter (PH promoter), polyhedrin-coding residues (phcr), and multiple cloning site in Ac-omega are acquired from the transfer vector pVL1393. (B) The nucleotide sequence of the AcMNPV derivative Ac-omega at the I-Sce I recognition site, including the polyhedrin promoter region and the 5′ and 3′ phcr. The arrows (positions -57 and -48) indicate the 5′ end of the mRNA and the transcription start site, respectively. Natural polyhedrin ATG is mutated to ATT (+1, underlined). The lowercase letters (positions +1 to +35) indicate 5′ phcr. The 18-bp I-Sce I recognition site spans from nucleotides +45 to +62 (underlined). The I-Sce I 3′ nonpalindromic overhang of four bases is also shown (bold). The target-gene insertion site (unidirectional only) is located at position +58. Nucleotides +65 to +110 are derived from the multiple cloning site of transfer vector pVL1393. At position +111, the 3′ polyhedrin-coding region continues with nucleotide +172 of the natural polyhedrin gene (lowercase).
We are grateful to Professor Loy Volkman of the University of California at Berkeley for her valuable contributions. Previous Section