Vector For Expression Of Foreign Genes Research Articles

Evaluation of the genotype I Japanese encephalitis virus as a stable viral vector for foreign gene expression

Manipulation of the flavivirus genome to accommodate and express a heterologous gene of interest has become an attractive approach for gene delivery and the development of viral-vectored vaccines. However, due to the inherent genetic instability of the flavivirus genomes, the construction of recombinant viruses carrying a foreign gene could be problematic and heavily resistant. In this study, the possibility of the Japanese encephalitis virus (JEV) as a stable flavivirus vector for the expression of a foreign gene was assessed using reverse genetics. The full-length cDNA genome of genotype I (GI) JEV inherently possessed excellent stability and manipulability in a bacterial host, while mutations and deletions accumulated in the cDNA genomes of genotype Ⅲ (GⅢ) JEV strains. Using the GI JEV as backbones, we generate a panel of recombinant viruses expressing various foreign genes. All recombinant viruses exhibited excellent genetic stability and efficiently express foreign genes for at least ten serial passages in vitro. In application, a convenient, rapid and reliable image-based assay for neutralizing antibody testing and antiviral drug discovery was established with a mCherry-reporter recombinant virus (rBJ-mCherry). Meanwhile, the recombinant viruses expressing the antigens of the African swine fever virus (ASFV) or Classical swine fever virus (CSFV) could effectively induce antibody responses to the JEV vector and foreign antigens in a mouse vaccination model. Therefore, GI JEV strains could serve as viral vectors accommodating the expression of large foreign genes.

Construction and characterization of a full-length infectious clone of Getah virus in vivo.

Getah virus (GETV) is a mosquito-borne virus of the genus Alphavirus in the family Togaviridae and, in recent years, it has caused several outbreaks in animals. The molecular basis for GETV pathogenicity is not well understood. Therefore, a reverse genetic system of GETV is needed to produce genetically modified viruses for the study of the viral replication and its pathogenic mechanism. Here, we generated a CMV-driven infectious cDNA clone based on a previously isolated GETV strain, GX201808 (pGETV-GX). Transfection of pGETV-GX into BHK-21 ​cells resulted in the recovery of a recombinant virus (rGETV-GX) which showed similar growth characteristics to its parental virus. Then three-day-old mice were experimentally infected with either the parental or recombinant virus. The recombinant virus showed milder pathogenicity than the parental virus in the mice. Based on the established CMV-driven cDNA clone, subgenomic promoter and two restriction enzyme sites (BamHI and EcoRI) were introduced into the region between E1 protein and 3′UTR. Then the green fluorescent protein (GFP), red fluorescent protein (RFP) and improved light-oxygen-voltage (iLOV) genes were inserted into the restriction enzyme sites. Transfection of the constructs carrying the reporter genes into BHK-21 ​cells proved the rescue of the recombinant reporter viruses. Taken together, the establishment of a reverse genetic system for GETV provides a valuable tool for the study of the virus life cycle, and to aid the development of genetically engineered GETVs as vectors for foreign gene expression.

The infectious cDNA clone of commercial HP-PRRS JXA1-R-attenuated vaccine can be a potential effective live vaccine vector.

Multiple commercial porcine reproductive and respiratory syndrome (PRRS) modified live vaccines are currently utilized in Chinese swine herds due to the limited cross-protection of vaccines and coexistence of different PRRS viruses. In this study, an infectious cDNA clone of the highly pathogenic PRRS (HP-PRRS) vaccine JXA1-R strain was generated. We successfully rescued the virus from direct in vitro DNA transfection of rJXA1-R clone, which has similar growth kinetics to the parental JXA1-R virus in Marc-145 cells. To further evaluate the potential use of the cloned rJXA1-R virus as a live vector for foreign gene expression, the enhanced green fluorescent protein (EGFP) was inserted between non-structural and structural genes. Our results showed that the dynamic expression of EGFP can be visualized by live cell imaging system during the infection in Marc-145 cells. The availability of our cloned JXA1-R viruses provides a crucial platform to study the fundamental biology of HP-PRRS virus vaccine and also serves as a potential effective vector for developing live vector vaccines against swine pathogens.

A Foxtail mosaic virus Vector for Virus-Induced Gene Silencing in Maize.

Plant viruses have been widely used as vectors for foreign gene expression and virus-induced gene silencing (VIGS). A limited number of viruses have been developed into viral vectors for the purposes of gene expression or VIGS in monocotyledonous plants, and among these, the tripartite viruses Brome mosaic virus and Cucumber mosaic virus have been shown to induce VIGS in maize (Zea mays). We describe here a new DNA-based VIGS system derived from Foxtail mosaic virus (FoMV), a monopartite virus that is able to establish systemic infection and silencing of endogenous maize genes homologous to gene fragments inserted into the FoMV genome. To demonstrate VIGS applications of this FoMV vector system, four genes, phytoene desaturase (functions in carotenoid biosynthesis), lesion mimic22 (encodes a key enzyme of the porphyrin pathway), iojap (functions in plastid development), and brown midrib3 (caffeic acid O-methyltransferase), were silenced and characterized in the sweet corn line Golden × Bantam. Furthermore, we demonstrate that the FoMV infectious clone establishes systemic infection in maize inbred lines, sorghum (Sorghum bicolor), and green foxtail (Setaria viridis), indicating the potential wide applications of this viral vector system for functional genomics studies in maize and other monocots.

Development of tobacco ringspot virus-based vectors for foreign gene expression and virus-induced gene silencing in a variety of plants

We report here the development of tobacco ringspot virus (TRSV)-based vectors for the transient expression of foreign genes and for the analysis of endogenous gene function in plants using virus-induced gene silencing. The jellyfish green fluorescent protein (GFP) gene was inserted between the TRSV movement protein (MP) and coat protein (CP) regions, resulting in high in-frame expression of the RNA2-encoded viral polyprotein. GFP was released from the polyprotein via an N-terminal homologous MP-CP cleavage site and a C-terminal foot-and-mouth disease virus (FMDV) 2 A catalytic peptide in Nicotiana benthamiana. The VIGS target gene was introduced in the sense and antisense orientations into a SnaBI site, which was created by mutating the sequence following the CP stop codon. VIGS of phytoene desaturase (PDS) in N. benthamiana, Arabidopsis ecotype Col-0, cucurbits and legumes led to obvious photo-bleaching phenotypes. A significant reduction in PDS mRNA levels in silenced plants was confirmed by semi-quantitative RT-PCR.

P and M gene junction is the optimal insertion site in Newcastle disease virus vaccine vector for foreign gene expression

Newcastle disease virus (NDV) has been developed as a vector for vaccine and gene therapy purposes. However, the optimal insertion site for foreign gene expression remained to be determined. In the present study, we inserted the green fluorescence protein (GFP) gene into five different intergenic regions of the enterotropic NDV VG/GA vaccine strain using reverse genetics technology. The rescued recombinant viruses retained lentogenic pathotype and displayed delayed growth dynamics, particularly when the GFP gene was inserted between the NP and P genes of the virus. The GFP mRNA level was most abundant when the gene was inserted closer to the 3' end and gradually decreased as the gene was inserted closer to the 5' end. Measurement of the GFP fluorescence intensity in recombinant virus-infected cells demonstrated that the non-coding region between the P and M genes is the optimal insertion site for foreign gene expression in the VG/GA vaccine vector.

Improving initial infectivity of the Turnip mosaic virus (TuMV) infectious clone by an mini binary vector via agro-infiltration.

BackgroundThe in vivo infectious clone of Turnip mosaic virus (TuMV), p35S-TuMV, was used on plant pathology research for many years. To activate p35S-TuMV, the plasmid was mechanically introduced to the local lesion host Chenopodium quinoa. However, low infectivity occurred when the TuMV from C. quinoa was transferred to the systemic host Nicotiana benthamiana.ResultsTo increase the efficiency of initial infectivity on N. benthamiana, the expression of the TuMV infectious clone by a binary vector that directly activates viral RNA through agro-infiltration is considered to be a good alternative. The size of the binary vector by agro-infiltration is usually large and its backbone has numerous restriction sites that increase difficulty for construction. In this study, we attempted to construct a mini binary vector (pBD003) with less restriction sites. The full-length cDNA of TuMV genome, with or without green fluorescence protein, was inserted in pBD003 to generate pBD-TuMV constructs, which were then individually introduced to N. benthamiana plants by agro-infiltration. Symptom development and ELISA positivity with TuMV antiserum indicated that the pBD-TuMV constructs are infectious. Moreover, the initial infectivity of a mild strain TuMV-GK, which contains an R182K mutation on HC-Pro, constructed in the pBD003 vector was significantly increased by agro-infiltration.ConclusionThus, we concluded that the newly constructed mini binary vector provides a more feasible tool for TuMV researches in areas, such as creating a mild strain for cross-protection, or a viral vector for foreign gene expression. In addition, the multiple cloning sites will be further cloned in pBD003 for convenience in constructing other viral infectious clones.Electronic supplementary materialThe online version of this article (doi:10.1186/1999-3110-54-22) contains supplementary material, which is available to authorized users.

Rana grylio virus as a vector for foreign gene expression in fish cells

In the present study, Rana grylio virus (RGV, an iridovirus) thymidine kinase (TK) gene and viral envelope protein 53R gene were chosen as targets for foreign gene insertion. ΔTK-RGV and Δ53R-RGV, two recombinant RGV, expressing enhanced green fluorescence protein (EGFP) were constructed and analyzed in Epithelioma papulosum cyprinid (EPC) cells. The EGFP gene which fused to the virus major capsid protein (MCP) promoter p50 was inserted into TK and 53R gene loci of RGV, respectively. Cells infected with these two recombinant viruses not only displayed plaques, but also emitted strong green fluorescence under fluorescence microscope, providing a simple method for selection and purification of recombinant viruses. ΔTK-RGV was purified by seven successive rounds of plaque isolation and could be stably propagated in EPC cells. All of the plaques produced by the purified recombinant virus emitted green fluorescence. However, Δ53R-RGV was hard to be purified even through twenty rounds of plaque isolation. The purified recombinant virus ΔTK-RGV was verified by PCR analysis and Western blotting. These results showed EGFP was expressed in ΔTK-RGV infected cells. Furthermore, one-step growth curves and electron microscopy revealed that infection with recombinant ΔTK-RGV and wild-type RGV are similar. Therefore, RGV was demonstrated could be as a viral vector for foreign gene expression in fish cells.

Production of viral vectors using recombinase-mediated cassette exchange

DNA viruses are often used as vectors for foreign gene expression, but large DNA region from cloned or authentic viral genomes must usually be handled to generate viral vectors. Here, we present a unique system for generating adenoviral vectors by directly substituting a gene of interest in a small transfected plasmid with a replaced gene in a replicating viral genome in Cre-expressing 293 cells using the recombinase-mediated cassette exchange (RMCE) reaction. In combination with a positive selection of the viral cis-acting packaging signal connected with the gene of interest, the purpose vector was enriched to 97.5 and 99.8% after three and four cycles of infection, respectively. Our results also showed that the mutant loxP V (previously called loxP 2272), a variant target of Cre used in the RMCE reaction, was useful as a non-compatible mutant to wild-type loxP. This method could be useful for generating not only a large number of adenovirus vectors simultaneously, but also other DNA virus vectors including helper-dependent adenovirus vector.

Virus promoters determine interference by defective RNAs: selective amplification of mini-RNA vectors and rescue from cDNA by a 3' copy-back ambisense rabies virus.

Typical defective interfering (DI) RNAs are more successful in the competition for viral polymerase than the parental (helper) virus, which is mostly due to an altered DI promoter composition. Rabies virus (RV) internal deletion RNAs which possess the authentic RV terminal promoters, and which therefore are transcriptionally active and can be used as vectors for foreign gene expression, are poorly propagated in RV-infected cells and do not interfere with RV replication. To allow DI-like amplification and high-level gene expression from such mini-RNA vectors, we have used an engineered 3' copy-back (ambisense) helper RV in which the strong replication promoter of the antigenome was replaced with the 50-fold-weaker genome promoter. In cells coinfected with ambisense helper virus and mini-RNAs encoding chloramphenicol acetyltransferase (CAT) and luciferase, mini-RNAs were amplified to high levels. This was correlated with interference with helper virus replication, finally resulting in a clear predominance of mini-RNAs over helper virus. However, efficient successive passaging of mini-RNAs and high-level reporter gene activity could be achieved without adding exogenous helper virus, revealing a rather moderate degree of interference not precluding substantial HV propagation. Compared to infections with recombinant RV vectors expressing CAT, the availability of abundant mini-RNA templates led to increased levels of CAT mRNA such that CAT activities were augmented up to 250-fold, while virus gene transcription was kept to a minimum. We have also exploited the finding that internal deletion model RNAs behave like DI RNAs and are selectively amplified in the presence of ambisense helper virus to demonstrate for the first time RV-supported rescue of cDNA after transfection of mini-RNA cDNAs in ambisense RV-infected cells expressing T7 RNA polymerase.

Plant virus gene vectors for transient expression of foreign proteins in plants.

The development of plant virus gene vectors for expression of foreign genes in plants provides attractive biotechnological tools to complement conventional breeding and transgenic methodology. The benefits of virus-based transient RNA and DNA replicons versus transgenic gene expression include rapid and convenient engineering coupled with flexibility for expeditious application in various plant species. These characteristics are especially advantageous when very high levels of gene expression are desired within a short time, although instability of the foreign gene in the viral genome can present some problems. The strategies that have been tested for foreign gene expression in various virus-based vectors include gene replacement, gene insertion, epitope presentation, use of virus controlled gene expression cassettes, and complementation. Recent reports of the utilization of virus vectors for foreign gene expression in fundamental research and biotechnology applications are discussed.

Immediate-Early Baculovirus Vectors for Foreign Gene Expression in Transformed or Infected Insect Cells

Baculovirus expression vectors are used routinely for foreign gene expression and are under intense development as improved biological pesticides. Conventional baculovirus expression vectors are recombinant viruses that can express a foreign gene in insect cells under the control of thepolyhedrinpromoter, which provides high-level transcription during the very late phase of infection. For some applications, including foreign glycoprotein production and insect pest control, it might be advantageous to have baculovirus vectors that could express foreign gene products in uninfected cells or earlier after infection. To fulfill this need, we have constructed a new set of plasmids that can be used to clone and express foreign genes under the control of a baculovirusie1promoter, which is active in uninfected insect cells and throughout infection. We used a subset of these new plasmids to isolate recombinant baculoviruses containing various foreign genes and compared expression of these genes by the resulting immediate-early baculovirus vectors and by conventional baculovirus vectors. As expected, the immediate-early vectors began to express each foreign gene earlier in infection but, by 36–48 h postinfection, the conventional vectors had produced more of each foreign protein. Conventional baculovirus vectors also produced more enzymatic activity from two different procaryotic genes than the immediate–early baculovirus vectors. However, immediate-early vectors produced as much or more enzymatic activity from two different eucaryotic genes encoding secretory pathway proteins than the conventional vectors, even at 48 h postinfection. Hence, this report describes a new set of plasmids that can be used to clone and express foreign genes under the control of the baculovirusie1promoter and suggests that immediate-early baculovirus vectors might be as useful as conventional baculovirus expression vectors for producing biologically active eucaryotic secretory pathway proteins.

Replication of subterranean clover stunt virus in pea and subterranean clover protoplasts

Subterranean clover stunt virus (SCSV) was previously found to be representative of a new type of single-stranded DNA virus. Purified SCSV particles did not infect subterranean clover when various attempts were made to transmit them to this host using aphids ( Aphis craccivora) which had previously been fed on preparations of the virus. To demonstrate that purified preparations of SCSV are capable of replication, pea and subterranean clover protoplasts were inoculated with the virus using PEG treatment or electroporation, and maintained for up to 13 days. Up to 40% of the protoplasts survived after PEG treatment but less than 10% survived after electroporation. Both enzyme-linked immunosorbent assay (ELISA) and nucleic acid hybridisation detected de novo synthesis of SCSV in the inoculated protoplasts. The amount of SCSV coat protein detected by ELISA decreased from day 0 to day 3 post-inoculation but increased thereafter over several days to a maximum at about 10 days. Nucleic acid hybridisation studies using strand-specific probes showed that the kinetics of synthesis of the virion and its complementary strand nucleic acid corresponded with the kinetics of accumulation of SCSV antigens. These results suggest that purified SCSV particles are capable of replication in transient protoplast systems. The protoplast assay could be used to characterise new or altered segments or functional domains of the SCSV genome and for the development of SCSV genome as a vector for foreign gene expression in plants.

Expression of the Autographa californica nuclear polyhedrosis virus p10 gene: effect of polyhedrin gene expression.

Two major late proteins, polyhedrin and p10, are synthesized in large quantities in baculovirus infected insect cells. This and the fact that both proteins are dispensable for virus replication, form the basis for the use of these viruses as vector for foreign gene expression. To address the question whether the Autographa californica nuclear polyhedrosis virus p10 promotor-driven expression is influenced by the concurrent expression of the polyhedrin gene, several recombinants were constructed with various deletions in the polyhedrin gene. The Escherichia coli lacZ gene was used as a marker to allow direct comparison between p10 and polyhedrin-driven expression. None of the deletions in the polyhedrin gene did result in higher expression of the p10 promoter-controlled gene. This suggested that the transcriptional and/or translational activity of the p10 and polyhedrin gene are independently regulated. To compare the level of polyhedrin and p10 promoter driven expression, recombinants with the lacZ gene cloned behind either promoter were studied. No significant difference in level of expression was observed. In cells infected with a recombinant with the lacZ gene present behind both promoters a reduced level of expression was observed, whereas a considerable increase was expected. This may be due to instability of the viral genome, as two copies of the lacZ gene were present.

Baculoviruses as vectors for foreign gene expression in insect cells

AbstractRecombinant DNA technology has enabled the expression of a large number of eukaryotic genes in vitro. There has been growing interest in recent years in the development of baculovirus expression vectors as an easily manipulated gene expression system. Both virus and insect cell culture are relatively easy to handle, and biologically active proteins have been produced abundantly from a variety of eukaryotic, prokaryotic and viral genes. This review describes the molecular biology of baculoviruses and some of the current applications of the baculovirus expression system in insect cells. Specific emphasis is placed on those features relevant to the use of this system in pesticide research.

59] Yeast vectors for production of interferon

This chapter describes the use of two different yeast promoters to direct the synthesis of interferon (IFN)-α and IFN-γ. The yeast Saccharomyces cerevisae has been used for hundreds of years as an industrial microorganism, having proven its utility in both brewing and baking. Yeast has been utilized in the laboratory for many decades as a model system for the study of many cellular processes. Since the development of molecular cloning techniques in microorganisms, yeast has become increasingly popular for the study of heterologous gene expression. As a eukaryote, yeast cells potentially possess much of the cellular machinery necessary for the expression of foreign eukaryotic genes and for the proper modification of the gene product. Yeast vectors for foreign gene expression contain certain requisite features. The elements of the vector are generally derived from yeast and bacteria to permit the propagation of the plasmid in both. Because APase is a secreted enzyme in yeast, the pYE71 vectors with IFN-αA and IFN-γ might be expected to direct secretion of interferon, but little or no activity is detected in media from induced cells carrying these plasmids. However, there is evidence that the presence of the signal peptide does effect the cellular location of the interferon.