ABSTRACT
Baculoviruses can express transgenes in a wide range of vertebrate cells. However, in some cells transgene expression is weak. To enhance transgene expression, we studied the effect of the Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) on baculovirus (BV)-mediated gene expression of several transgenes. A significant increase in BV-mediated gene expression was detected in several cell lines. A 10-fold increase in transgene expression was observed with the WPRE as determined by the percentage of positive cells and mean fluorescence intensity (MFI). Furthermore, a combination of optimized cell culture medium and WPRE virus led to more than a 60-fold increase in gene expression. In accordance, elevated mRNA and protein levels were detected in WPRE-virus transduced cells. In HepG2 and RaaSMC, WPRE-mediated enhancement was comparable to the previously shown positive effect of sodium butyrate on BV-mediated gene expression. Thus, inclusion of the WPRE into a baculovirus vector provides a simple means to improve BV-mediated gene expression in vertebrate cells.
Subject(s)
Baculoviridae/genetics , Baculoviridae/metabolism , Gene Expression , Regulatory Sequences, Nucleic Acid/genetics , Transgenes/genetics , Animals , Baculoviridae/drug effects , Butyrates/pharmacology , Cell Line , Flow Cytometry , Gene Expression/drug effects , Gene Expression Regulation/drug effects , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Hepatitis B Virus, Woodchuck , Humans , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rabbits , Transcription, Genetic/genetics , Transduction, Genetic , Vertebrates , beta-Galactosidase/metabolismABSTRACT
We have constructed a novel tetra-promoter vector (pBVboostFG) system that enables screening of gene/cDNA libraries for functional genomic studies. The vector enables an all-in-one strategy for gene expression in mammalian, bacterial and insect cells and is also suitable for direct use in vivo. Virus preparation is based on an improved mini Tn7 transpositional system allowing easy and fast production of recombinant baculoviruses with high diversity and negligible background. Cloning of the desired DNA fragments or libraries is based on the recombination system of bacteriophage lambda. As an example of the utility of the vector, genes or cDNAs of 18 different proteins were cloned into pBVboostFG and expressed in different hosts. As a proof-of-principle of using the vector for library screening, a chromophoric Thr65-Tyr-Gly67-stretch of enhanced green fluorescent protein was destroyed and subsequently restored by novel PCR strategy and library screening. The pBVboostFG enables screening of genome-wide libraries, thus making it an efficient new platform technology for functional genomics.
Subject(s)
Baculoviridae/genetics , Escherichia coli/genetics , Gene Library , Genetic Vectors , Spodoptera/genetics , Animals , Bacteriophage lambda/genetics , Brain/metabolism , CHO Cells , Cell Line , Cricetinae , Cricetulus , Female , Gene Expression , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Rats , Rats, Wistar , Spodoptera/cytologyABSTRACT
An improved method for the generation of recombinant baculoviruses by Tn7-mediated transposition is described. The method is based on the modified donor vector (pBVboost) and an improved selection scheme of the baculovirus bacmids in Escherichia coli with a mutated SacB gene. Recombinant bacmids can be generated at a frequency of approximately 10(7)/microg of donor vector with a negligible background. This easy-to-use and efficient pBVboost system provides the basis for a high-throughput generation of recombinant baculoviruses as well as a more convenient way to produce single viruses. The introduced selection scheme is also useful for the construction of other vectors by transposition in E.coli.
