Strategy for generation of replication–competent recombinant rotaviruses expressing multiple foreign genes

2021 ◽  
Vol 102 (4) ◽  
Author(s):  
Riona Hatazawa ◽  
Saori Fukuda ◽  
Kanako Kumamoto ◽  
Fumio Matsushita ◽  
Shizuko Nagao ◽  
...  
Keyword(s):  
Reverse Genetics ◽  
Single Gene ◽  
Gene Segment ◽  
Reporter Genes ◽  
Expression Vectors ◽  
Foreign Gene ◽  
Reading Frame ◽  
Exogenous Genes ◽  
Foreign Genes ◽  
Recent Establishment

With the recent establishment of robust reverse genetics systems for rotavirus, rotavirus is being developed as a vector to express foreign genes. However, insertion of larger sequences such as those encoding multiple foreign genes into the rotavirus genome has been challenging because the virus segments are small. In this paper, we attempted to insert multiple foreign genes into a single gene segment of rotavirus to determine whether it can efficiently express multiple exogenous genes from its genome. At first, we engineered a truncated NSP1 segment platform lacking most of the NSP1 open reading frame and including a self-cleaving 2A sequence (2A), which made it possible to generate a recombinant rotavirus stably expressing NanoLuc (Nluc) luciferase as a model foreign gene. Based on this approach, we then demonstrated the generation of a replication-competent recombinant rotavirus expressing three reporter genes (Nluc, EGFP, and mCherry) by separating them with self-cleaving 2As, indicating the capacity of rotaviruses as to the insertion of multiple foreign genes. Importantly, the inserted multiple foreign genes remained genetically stable during serial passages in cell culture, indicating the potential of rotaviruses as attractive expression vectors. The strategy described here will serve as a model for the generation of rotavirus-based vectors designed for the expression and/or delivery of multiple foreign genes.

scholarly journals Coronaviruses as Vectors: Position Dependence of Foreign Gene Expression

2003 ◽  
Vol 77 (21) ◽  
pp. 11312-11323 ◽  
Author(s):  
Cornelis A. M. de Haan ◽  
Linda van Genne ◽  
Jeroen N. Stoop ◽  
Haukeline Volders ◽  
Peter J. M. Rottier
Keyword(s):  
Gene Expression ◽  
Expression Vectors ◽  
Luciferase Expression ◽  
Foreign Gene ◽  
Regulatory Sequences ◽  
Expression Levels ◽  
Genomic Position ◽  
Position Dependence ◽  
Foreign Genes

ABSTRACT Coronaviruses are the enveloped, positive-stranded RNA viruses with the largest RNA genomes known. Several features make these viruses attractive as vaccine and therapeutic vectors: (i) deletion of their nonessential genes is strongly attenuating; (ii) the genetic space thus created allows insertion of foreign information; and (iii) their tropism can be modified by manipulation of the viral spike. We studied here their ability to serve as expression vectors by inserting two different foreign genes and evaluating systematically the genomic position dependence of their expression, using a murine coronavirus as a model. Renilla and firefly luciferase expression cassettes, each provided with viral transcription regulatory sequences (TRSs), were inserted at several genomic positions, both independently in different viruses and combined within one viral genome. Recombinant viruses were generated by using a convenient method based on targeted recombination and host cell switching. In all cases high expression levels of the foreign genes were observed without severe effects on viral replication in vitro. The expression of the inserted gene appeared to be dependent on its genomic position, as well as on the identity of the gene. Expression levels increased when the luciferase gene was inserted closer to the 3′ end of the genome. The foreign gene insertions generally reduced the expression of upstream viral genes. The results are consistent with coronavirus transcription models in which the transcription from upstream TRSs is attenuated by downstream TRSs. Altogether, our observations clearly demonstrate the potential of coronaviruses as (multivalent) expression vectors.

scholarly journals Role of the 3′ untranslated region of baculovirus p10 mRNA in high-level expression of foreign genes

1999 ◽  
Vol 80 (8) ◽  
pp. 2253-2262 ◽  
Author(s):  
Monique M. van Oers ◽  
Just M. Vlak ◽  
Harry O. Voorma ◽  
Adri A. M. Thomas
Keyword(s):  
Gene Expression ◽  
Gene Cassette ◽  
Expression Vectors ◽  
Foreign Gene ◽  
Sequence Motif ◽  
Wild Type ◽  
Recombinant Viruses ◽  
Foreign Genes ◽  
High Level ◽  
Terminator Sequence

The p10 gene of Autographa californica nucleopolyhedrovirus has two putative AATAAA polyadenylation signals. The downstream signal is used predominantly, as was determined by analysing 3′ cDNA ends. This downstream motif is followed by a GT-rich sequence, known to be important for efficient polyadenylation in mammalian systems. To analyse the importance of polyadenylation for p10 gene expression, recombinant viruses with altered 3′ untranslated regions (UTRs) were tested using chloramphenicol acetyltransferase (CAT) as a reporter. Surprisingly, after inactivation of the downstream AATAAA motif, CAT expression remained at the same high level as observed with a wild-type 3′ UTR. Polyadenylation occurred 24–28 nucleotides further downstream, probably due to an ATTAAA sequence motif. Replacing the p10 3′ UTR with the SV40 early terminator sequence as part of an hsp70–lacZ–SV40 gene cassette, which is commonly used in baculovirus expression vectors, resulted in a reduction in reporter gene expression. Polyadenylation occurred far more efficiently in the original p10 3′ UTR than in the SV40 terminator sequence, as was shown by testing the SV40 terminator separately. These results indicate that in order to obtain high levels of foreign gene expression, vectors that provide a wild-type p10 3′ UTR are to be preferred over those containing the hsp70–lacZ–SV40 gene cassette. Comparison of the p10 genes of various baculoviruses showed the presence of at least one AATAAA or ATTAAA motif in combination with a GT-rich sequence in the 3′ UTR, suggesting an evolutionary conservation of these two elements, thereby maintaining the high level of p10 gene expression.

scholarly journals Development of Stable Rotavirus Reporter Expression Systems

2018 ◽  
Vol 93 (4) ◽  
Author(s):  
Yuta Kanai ◽  
Takahiro Kawagishi ◽  
Ryotaro Nouda ◽  
Misa Onishi ◽  
Pimfhun Pannacha ◽  
...  
Keyword(s):  
Amino Acids ◽  
Rna Structure ◽  
Reverse Genetics ◽  
Fluorescent Proteins ◽  
Reporter Genes ◽  
Chimeric Gene ◽  
Genome Replication ◽  
Reporter Protein ◽  
Reading Frame ◽  
Intestinal Pathogens

ABSTRACTEngineered recombinant viruses expressing reporter genes have been developed for real-time monitoring of replication and for mass screening of antiviral inhibitors. Recently, we reported using a reverse genetics system to develop the first recombinant reporter rotaviruses (RVs) that expressed NanoLuc (NLuc) luciferase. Here, we describe a strategy for developing stable reporter RVs expressing luciferase and green or red fluorescent proteins. The reporter genes were inserted into the open reading frame of NSP1 and expressed as a fusion with an NSP1 peptide consisting of amino acids 1 to 27. The stability of foreign genes within the reporter RV strains harboring a shorter chimeric NSP1-reporter gene was greater than that of those in the original reporter RV strain, independent of the transgene inserted. The improved reporter RV was used to screen for neutralizing monoclonal antibodies (MAbs). Sequence analysis of escape mutants from one MAb clone (clone 29) identified an amino acid substitution (arginine to glycine) at position 441 in the VP4 protein, which resides within neutralizing epitope 5-1 in the VP5* fragment. Furthermore, to express a native reporter protein lacking NSP1 amino acids 1 to 27, the 5′- and 3′-terminal region sequences were modified to restore the predicted secondary RNA structure of the NSP1-reporter chimeric gene. These data demonstrate the utility of reporter RVs for live monitoring of RV infections and also suggest further applications (e.g., RV vaccine vectors, which can induce mucosal immunity against intestinal pathogens).IMPORTANCEDevelopment of reporter RVs has been hampered by the lack of comprehensive reverse genetics systems. Recently, we developed a plasmid-based reverse genetics system that enables generation of reporter RVs expressing NLuc luciferase. The prototype reporter RV had some disadvantages (i.e., the transgene was unstable and was expressed as a fusion protein with a partial NSP1 peptide); however, the improved reporter RV overcomes these problems through modification of the untranslated region of the reporter-NSP1 chimeric gene. This strategy for generating stable reporter RVs could be expanded to diverse transgenes and be used to develop RV transduction vectors. Also, the data improve our understanding of the importance of 5′- and 3′-terminal sequences in terms of genome replication, assembly, and packaging.

scholarly journals Essential and Dispensable Virus-Encoded Replication Elements Revealed by Efforts To Develop Hypoviruses as Gene Expression Vectors

2000 ◽  
Vol 74 (16) ◽  
pp. 7568-7577 ◽  
Author(s):  
Nobuhiro Suzuki ◽  
Lynn M. Geletka ◽  
Donald L. Nuss
Keyword(s):  
Gene Expression ◽  
Fluorescent Protein ◽  
Cryphonectria Parasitica ◽  
Expression Vectors ◽  
Green Fluorescent Protein Gene ◽  
Reading Frame ◽  
Chestnut Blight Fungus ◽  
Virulence Attenuation ◽  
Foreign Genes ◽  
Green Fluorescent

ABSTRACT We have investigated whether hypoviruses, viral agents responsible for virulence attenuation (hypovirulence) of the chestnut blight fungusCryphonectria parasitica, could serve as gene expression vectors. The infectious cDNA clone of the prototypic hypovirus CHV1-EP713 was modified to generate 20 different vector candidates. Although transient expression was achieved for a subset of vectors that contained the green fluorescent protein gene from Aequorea victoria, long-term expression (past day 8) was not observed for any vector construct. Analysis of viral RNAs recovered from transfected fungal colonies revealed that the foreign genes were readily deleted from the replicating virus, although small portions of foreign sequences were retained by some vectors after months of replication. However, the results of vector viability and progeny characterization provided unexpected new insights into essential and dispensable elements of hypovirus replication. The N-terminal portion (codons 1 to 24) of the 5′-proximal open reading frame (ORF), ORF A, was found to be required for virus replication, while the remaining 598 codons of this ORF were completely dispensable. Substantial alterations were tolerated in the pentanucleotide UAAUG that contains the ORF A termination codon and the overlapping putative initiation codon of the second of the two hypovirus ORFs, ORF B. Replication competence was maintained following either a frameshift mutation that caused a two-codon extension of ORF A or a modification that produced a single-ORF genomic organization. These results are discussed in terms of determinants of hypovirus replication, the potential utility of hypoviruses as gene expression vectors, and possible mechanisms by which hypoviruses recognize and delete foreign sequences.

scholarly journals Dual Reproductive Cell-Specific Promoter-Mediated Split-Cre/LoxP System Suitable for Exogenous Gene Deletion in Hybrid Progeny of Transgenic Arabidopsis

2021 ◽  
Vol 22 (10) ◽  
pp. 5080
Author(s):  
Chen Yang ◽  
Jia Ge ◽  
Xiaokang Fu ◽  
Keming Luo ◽  
Changzheng Xu
Keyword(s):  
Gene Deletion ◽  
Transgenic Arabidopsis ◽  
Reporter Genes ◽  
Gm Crops ◽  
Expression Vectors ◽  
Reproductive Cell ◽  
Split Intein ◽  
F1 Progeny ◽  
Transgenic Lines ◽  
Exogenous Genes

Genetically modified (GM) crops possess some superior characteristics, such as high yield and insect resistance, but their biosafety has aroused broad public concern. Some genetic engineering technologies have recently been proposed to remove exogenous genes from GM crops. Few approaches have been applied to maintain advantageous traits, but excising exogenous genes in seeds or fruits from these hybrid crops has led to the generation of harvested food without exogenous genes. In a previous study, split-Cre mediated by split intein could recombine its structure and restore recombination activity in hybrid plants. In the current study, the recombination efficiency of split-Cre under the control of ovule-specific or pollen-specific promoters was validated by hybridization of transgenic Arabidopsis containing the improved expression vectors. In these vectors, all exogenous genes were flanked by two loxP sites, including promoters, resistance genes, reporter genes, and split-Cre genes linked to the reporter genes via LP4/2A. A gene deletion system was designed in which NCre was driven by proDD45, and CCre was driven by proACA9 and proDLL. Transgenic lines containing NCre were used as paternal lines to hybridize with transgenic lines containing CCre. Because this hybridization method results in no co-expression of the NCre and CCre genes controlled by reproduction-specific promoters in the F1 progeny, the desirable characteristics could be retained. After self-crossing in F1 progeny, the expression level and protein activity of reporter genes were detected, and confirmed that recombination of split-Cre had occurred and the exogenous genes were partially deleted. The gene deletion efficiency represented by the quantitative measurements of GUS enzyme activity was over 59%, with the highest efficiency of 73% among variable hybrid combinations. Thus, in the present study a novel dual reproductive cell-specific promoter-mediated gene deletion system was developed that has the potential to take advantage of the merits of GM crops while alleviating biosafety concerns.

scholarly journals Engineering of Recombinant Sheep Pox Viruses Expressing Foreign Antigens

2021 ◽  
Vol 9 (5) ◽  
pp. 1005
Author(s):  
Olga Chervyakova ◽  
Elmira Tailakova ◽  
Nurlan Kozhabergenov ◽  
Sandugash Sadikaliyeva ◽  
Kulyaisan Sultankulova ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Viral Vector ◽  
Foot And Mouth Disease ◽  
Open Reading Frames ◽  
Foreign Gene ◽  
Mouth Disease Virus ◽  
Foreign Genes ◽  
Green Fluorescent ◽  
Reading Frames

Capripoxviruses with a host range limited to ruminants have the great potential to be used as vaccine vectors. The aim of this work was to evaluate attenuated sheep pox virus (SPPV) vaccine strain NISKHI as a vector expressing several genes. Open reading frames SPPV020 (ribonucleotide kinase) and SPPV066 (thymidine kinase) were selected as sites for the insertion of foreign genes. Two integration plasmids with expression cassette were designed and constructed. Recombinant SPPVs expressing an enhanced green fluorescent protein (EGFP) (rSPPV(RRΔ)EGFP and rSPPV(TKΔ)EGFP), Foot-and-mouth disease virus capsid protein (VP1), and Brucella spp. outer membrane protein 25 (OMP25) (rSPPV(RRΔ)VP1A-(TKΔ)OMP25) were generated under the transient dominant selection method. The insertion of foreign genes into the SPPV020 and SPPV066 open reading frames did not influence the replication of the recombinant viruses in the cells. Successful foreign gene expression in vitro was assessed by luminescent microscopy (EGFP) and Western blot (VP1 and OMP25). Our results have shown that foreign genes were expressed by rSPPV both in permissive (lamb testicles) and non-permissive (bovine kidney, saiga kidney, porcine kidney) cells. Mice immunized with rSPPV(RRΔ)VP1A-(TKΔ)OMP25 elicited specific antibodies to both SPPV and foreign genes VP1 and OMP25. Thus, SPPV NISKHI may be used as a potential safe immunogenic viral vector for the development of polyvalent vaccines.

scholarly journals Cosegregation of Single Genes Associated with Fertility Restoration and Transcript Processing of Sorghum Mitochondrial orf107 and urf209

Genetics ◽  
1998 ◽  
Vol 150 (1) ◽  
pp. 383-391 ◽  
Author(s):  
Hoang V Tang ◽  
Ruying Chang ◽  
Daryl R Pring
Keyword(s):  
Fertility Restoration ◽  
Mitochondrial Gene ◽  
Pollen Viability ◽  
Single Gene ◽  
Dominant Gene ◽  
Nuclear Gene ◽  
Male Sterile ◽  
Reading Frame ◽  
Transcript Processing ◽  
Backcross Populations

Abstract Defective nuclear-cytoplasmic interactions leading to aberrant microgametogenesis in sorghum carrying the IS1112C male-sterile cytoplasm occur very late in pollen maturation. Amelioration of this condition, the restoration of pollen viability, involves a novel two-gene gametophytic system, wherein genes designated Rf3 and Rf4 are required for viability of individual gametes. Rf3 is tightly linked to, or represents, a single gene that regulates a transcript processing activity that cleaves transcriptsof orf107, a chimeric mitochondrial open reading frame specific to IS1112C. The mitochondrial gene urf 209 is also subject to nucleus-specific enhanced transcript processing, 5′ to the gene, conferred by a single dominant gene designated Mmt1. Examinations of transcript patterns in F2 and two backcross populations indicated cosegregation of the augmented orf107 and urf209 processing activities in IS1112C. Several sorghum lines that do not restore fertility or confer orf107 transcript processing do exhibit urf209 transcript processing, indicating that the activities are distinguishable. We conclude that the nuclear gene(s) conferring enhanced orf107 and urf209 processing activities are tightly linked in IS1112C. Alternatively, the similarity in apparent regulatory action of the genes may indicate allelic differences wherein the IS1112C Rf3 allele may differ from alleles of maintainer lines by the capability to regulate both orf107 and urf209 processing activities.

scholarly journals Coronaviruses as Vectors: Stability of Foreign Gene Expression

2005 ◽  
Vol 79 (20) ◽  
pp. 12742-12751 ◽  
Author(s):  
Cornelis A. M. de Haan ◽  
Bert Jan Haijema ◽  
David Boss ◽  
Frank W. H. Heuts ◽  
Peter J. M. Rottier
Keyword(s):  
Genetic Stability ◽  
Vaccine Development ◽  
Hepatitis Virus ◽  
Firefly Luciferase ◽  
Foreign Gene ◽  
Rna Recombination ◽  
Large Deletions ◽  
Group 2 ◽  
Attenuated Viruses ◽  
Foreign Genes

ABSTRACT Coronaviruses are enveloped, positive-stranded RNA viruses considered to be promising vectors for vaccine development, as (i) genes can be deleted, resulting in attenuated viruses; (ii) their tropism can be modified by manipulation of their spike protein; and (iii) heterologous genes can be expressed by simply inserting them with appropriate coronaviral transcription signals into the genome. For any live vector, genetic stability is an essential requirement. However, little is known about the genetic stability of recombinant coronaviruses expressing foreign genes. In this study, the Renilla and the firefly luciferase genes were systematically analyzed for their stability after insertion at various genomic positions in the group 1 coronavirus feline infectious peritonitis virus and in the group 2 coronavirus mouse hepatitis virus. It appeared that the two genes exhibit intrinsic differences, the Renilla gene consistently being maintained more stably than the firefly gene. This difference was not caused by genome size restrictions, by different effects of the encoded proteins, or by different consequences of the synthesis of the additional subgenomic mRNAs. The loss of expression of the firefly luciferase was found to result from various, often large deletions of the gene, probably due to RNA recombination. The extent of this process appeared to depend strongly on the coronaviral genomic background, the luciferase gene being much more stable in the feline than in the mouse coronavirus genome. It also depended significantly on the particular genomic location at which the gene was inserted. The data indicate that foreign sequences are more stably maintained when replacing nonessential coronaviral genes.

scholarly journals Selectable Markers and Reporter Genes for Engineering the Chloroplast of Chlamydomonas reinhardtii

Biology ◽  
2018 ◽  
Vol 7 (4) ◽  
pp. 46 ◽  
Author(s):  
Lola Esland ◽  
Marco Larrea-Alvarez ◽  
Saul Purton
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Higher Plants ◽  
Reporter Genes ◽  
Bioactive Metabolites ◽  
Cell Protein ◽  
Cell Factory ◽  
Expression Platform ◽  
Foreign Genes ◽  
Valuable Compounds ◽  
High Level

Chlamydomonas reinhardtii is a model alga of increasing interest as a cell factory for the production of valuable compounds, including therapeutic proteins and bioactive metabolites. Expression of foreign genes in the chloroplast is particularly advantageous as: (i) accumulation of product in this sub-cellular compartment minimises potential toxicity to the rest of the cell; (ii) genes can integrate at specific loci of the chloroplast genome (plastome) by homologous recombination; (iii) the high ploidy of the plastome and the high-level expression of chloroplast genes can be exploited to achieve levels of recombinant protein as high as 5% total cell protein; (iv) the lack of any gene silencing mechanisms in the chloroplast ensures stable expression of transgenes. However, the generation of C. reinhardtii chloroplast transformants requires efficient methods of selection, and ideally methods for subsequent marker removal. Additionally, the use of reporter genes is critical to achieving a comprehensive understanding of gene expression, thereby informing experimental design for recombinant applications. This review discusses currently available selection and reporter systems for chloroplast engineering in C. reinhardtii, as well as those used for chloroplast engineering in higher plants and other microalgae, and looks to the future in terms of possible new markers and reporters that will further advance the C. reinhardtii chloroplast as an expression platform.

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