Foreign Genes In Insect Cells Research Articles

Transient Expression of Foreign Genes in Insect Cells (sf9) for Protein Functional Assay

The transient gene expression system is one of the most important technologies for performing protein functional analysis in the baculovirus in vitro cell culture system. This system was developed to express foreign genes under the control of the baculoviral promoter in transient expression plasmids. Furthermore, this system can be applied to a functional assay of either the baculovirus itself or foreign proteins. The most widely and commercially available transient gene expression system is developed based on the immediate-early gene (IE) promoter of Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV). However, a low expression level of foreign genes in insect cells was observed. Therefore, a transient gene expression system was constructed for improving protein expression. In this system, recombinant plasmids were constructed to contain the target sequence under the control of the Drosophila heat shock 70 (Dhsp70) promoter. This protocol presents the application of this heat shock-based pDHsp/V5-His (V5 epitope with 6 histidine)/Spodoptera frugiperda cell (sf9 cell) system; this system is available not only for gene expression but also for evaluating the anti-apoptotic activity of candidate proteins in insect cells. Furthermore, this system can be either transfected with one recombinant plasmid or co-transfected two potentially functionally antagonistic recombinant plasmids in insect cells. The protocol demonstrates the efficiency of this system and provides a practical case of this technique.

Encyclopedia of Autographa californica nucleopolyhedrovirus genes

The Autographa californica multiple capsid nucleopolyhedrovirus (AcMNPV) was the first baculovirus for which the complete nucleotide sequence became known. Since then 15 years lapsed and much research has been performed to elucidate putative functions of the annotated open reading frames of this virus and this endeavour is still ongoing. AcMNPV is the most well-known and well-studied baculovirus species, not in the least for its application as a vector for the high-level expression of foreign genes in insect cells. This article is the first monograph of a single baculovirus and gives a current overview of what is known about the 151 AcMNPV ORFs, including (putative) function and temporal and spatial presence of transcripts and protein. To date 60 ORFs have a proven function, another 19 ORFs have homologs for which functions are known in other baculoviruses and 72 ORFs are still enigmatic. This paper should assist the reader in quickly finding the essentials of AcMNPV.

Tight transcriptional regulation of foreign genes in insect cells using an ecdysone receptor-based inducible system

The use of insect cells has been highly successful for the expression of foreign proteins from baculoviruses or plasmid vectors. Here, we describe a tight transcriptional regulation of foreign genes in insect cells using an ecdysone receptor-based inducible system. The system includes the DEF domains of the spruce budworm (Choristoneura fumiferana) EcR (CfEcR) fused to the Saccharomyces cerevisiae GAL4 DNA-binding domain and the EF domains of mammalian Mus musculus retinoid X receptor (MmRXR) fused to the acidic activation domains (AADs) of the baculovirus transactivators IE1 and IE0. Using a GAL4 response element in reporter constructs, both transient and stable expression in insect lepidopteran cells showed that the chimeric MmRXR and CfEcR only activated the reporter genes in the presence of inducer; no gene expression was detectable in the absence of inducer. Characterization of heterogenous activation domains in insect cells showed that the AADs from Autographa californica multiple nucleopolyhedrovirus (MNPV) IE1 and Orgyia pseudotsugata MNPV IE0 consistently exhibited higher inducible levels than the archetype AAD from herpesvirus VP16 in insect cells. To confirm the tight regulation of this system the highly toxic protein, diphtheria toxin (DT), was used. In the absence of an inducer no cytotoxic effect was observed in insect cells that had been transiently transformed with DT expressing plasmids. This system will therefore be a very useful tool for biotechnology applications expressing highly toxic proteins in insect cells and for studying the functional genomics of insects and microorganisms that infect them.

Baculovirus-mediated gene transfer into pancreatic islet cells.

Baculovirus transduction is a gene transfer method that uses a moth cell virus for mammalian cells in culture, which results in a high-level prolonged expression. Here we demonstrate that recombinant baculoviruses can serve as efficient gene transfer vehicles for delivering foreign genes driven by mammalian promoters into human and mouse pancreatic islet cells. Existing methods, such as various transfection and electroporation techniques, either suffer from low efficiency or cause extensive membrane damage. Viral vectors have emerged as an important tool for gene delivery and expression in mammalian cells but suffer from several drawbacks, such as lengthy construction time and expression of viral genes. The baculovirus Autographa californica multiple nuclear polyhedrosis virus is widely used as a vector for expression of foreign genes in insect cells and, more recently, in some mammalian cells. Using several green fluorescent protein- and LacZ-expressing constructs in a cytomegalovirus promoter cassette, we obtained efficient gene expression in primary human and mouse islet cells. There was no impairment of glucose-stimulated intracellular free calcium responses after baculovirus infection. The safety and the relative ease of construction and propagation of the virus makes the baculovirus system a useful tool for facilitating the transfer of foreign genes.

Autonomous Parvovirus and Densovirus Gene Vectors

Publisher Summary Viruses have evolved as efficient natural vehicles of genetic information and they are logical systems to develop as gene vectors. Several members of the family Parvoviridae and Densovirus are being evaluated for their potential as gene delivery vehicles. Recombinant MVM and LuIII vectors bearing reporter genes have been used to transduce various transformed or untransformed cell lines. To extend the utility of the LuIII vectors, LuIII luciferase transducing genomes have been pseudotyped with virions from heterologous parvoviruses. The results suggest that packaging LuIII transducing genomes into heterologous virions can effectively alter the specificity of transduction and may allow delivery of genes to a wider variety of cell types. When specific tissues are to be targeted, it should be possible to restrict expression of the transgene to certain cell or tissue types with the use of transcriptional control elements. In the case of densovirus, initial results with several densoviruses indicate that they and their promoters will be useful for introducing and expressing foreign genes in insect cells and probably live insects. The β-galactosidase gene has been inserted into genomes of both JcDNV and AeDNV, and then packaged into particles able to deliver ZacZ to target cells in culture, indicating that it will also be possible to utilize these vectors for gene delivery.

Efficient generation of infectious recombinant baculoviruses by site-specific transposon-mediated insertion of foreign genes into a baculovirus genome propagated in Escherichia coli.

The construction and purification of recombinant baculovirus vectors for the expression of foreign genes in insect cells by standard transfection and plaque assay methods can take as long as 4 to 6 weeks. This period can be reduced to several days by using a novel baculovirus shuttle vector (bacmid) that can replicate in Escherichia coli as a plasmid and can infect susceptible lepidopteran insect cells. The bacmid is a recombinant virus that contains a mini-F replicon, a kanamycin resistance marker, and attTn7, the target site for the bacterial transposon Tn7. Expression cassettes comprising a baculovirus promoter driving expression of a foreign gene that is flanked by the left and right ends of Tn7 can transpose to the target bacmid in E. coli when Tn7 transposition functions are provided in trans by a helper plasmid. The foreign gene is expressed when the resulting composite bacmid is introduced into insect cells.

The structural and functional organization of the Autographa californica nuclear polyhedrosis virus genome.

Baculoviruses are used as biological control agents of insect pests in agriculture and forestry. The multiple-nucleocapsid nuclear polyhedrosis virus of Autographa californica (AcMNPV) is the prototype baculovirus. Recently, this virus has become widely used as vector for the high-level expression of foreign genes in insect cells. An updated physical map of restriction sites as well as the location of open reading frames (ORFs) and transcripts are presented. Most characteristic is the dispersal of "early", "late", and "very late" genes over the genome and the presence of nested sets of 3' and 5' coterminal transcripts.

Trends in the Development of Baculovirus Expression Vectors

We describe the current status and potential use of baculovirus vectors for the expression of foreign genes in insect cells. Trends in the development of transfer vectors for the expression of foreign genes under the control of the strong polyhedrin promoter of Autographa californica nuclear polyhedrosis virus and strategies for maximizing levels of expression are discussed. Baculovirus vectors have achieved widespread acceptance for their ability to express proteins of agricultural and medical importance. A baculovirus vector was used to express the first recombinant HIV envelope proteins to receive F.D.A. approval for clinical evaluation as a candidate vaccine for AIDS. These insect DNA virus vectors are contributing to understanding the molecular biology of gene and protein function and regulation in both vertebrate and insect systems.