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 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.

The responsiveness of embryonic stem cells to alpha and beta interferons provides the basis of an inducible expression system for analysis of developmental control genes

1993 ◽  
Vol 13 (12) ◽  
pp. 7971-7976
Author(s):  
L M Whyatt ◽  
A Düwel ◽  
A G Smith ◽  
P D Rathjen
Keyword(s):  
Gene Expression ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Expression System ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Expression Vectors ◽  
Developmental Control ◽  
Developmental Potential

Embryonic stem (ES) cells, derived from the inner cell mass of the preimplantation mouse embryo, are used increasingly as an experimental tool for the investigation of early mammalian development. The differentiation of these cells in vitro can be used as an assay for factors that regulate early developmental decisions in the embryo, while the effects of altered gene expression during early embryogenesis can be analyzed in chimeric mice generated from modified ES cells. The experimental versatility of ES cells would be significantly increased by the development of systems which allow precise control of heterologous gene expression. In this paper, we report that ES cells are responsive to alpha and beta interferons (IFNs). This property has been exploited for the development of inducible ES cell expression vectors, using the promoter of the human IFN-inducible gene, 6-16. The properties of these vectors have been analyzed in both transiently and stably transfected ES cells. Expression was minimal or absent in unstimulated ES cells, could be stimulated up to 100-fold by treatment of the cells with IFN, and increased in linear fashion with increasing levels of IFN. High levels of induced expression were maintained for extended periods of time in the continuous presence of the inducing signal or following a 12-h pulse with IFN. Treatment of ES cells with IFN did not affect their growth or differentiation in vitro or compromise their developmental potential. This combination of features makes the 6-16-based expression vectors suitable for the functional analysis of developmental control control genes in ES cells.

scholarly journals Expression of mRNAs for Pro-and Anti-Apoptotic Factors in Granulosa Cells and Follicular Fluid of Women Undergoing in Vitro Fertilization. A Pilot Study

2021 ◽  
Author(s):  
Jozsef Bodis ◽  
Endre Sulyok ◽  
Akos Varnagy ◽  
Viktória Prémusz ◽  
Krisztina Godony ◽  
...  
Keyword(s):  
Gene Expression ◽  
In Vitro Fertilization ◽  
Mrna Expression ◽  
Granulosa Cells ◽  
Follicular Fluid ◽  
Vitro Fertilization ◽  
Expression Levels ◽  
The Impact ◽  
Apoptotic Factors

Abstract BackgroundThis observational clinical study evaluated the expression levels and predictive values of some apoptosis-related genes in granulosa cells (GCs) and follicular fluid (FF) of women undergoing in vitro fertilization (IVF).Methods GCs and FF were obtained at oocyte retrieval from 31 consecutive patients with heterogeneous infertility diagnosis (age: 34.3±5.8 years, body mass index: 24.02±3.12 kg/m2, duration of infertility: 4.2±2.1 years). mRNA expression of pro-apoptotic (BAX, CASP3, CASP8) and anti-apoptotic (BCL2, AMH, AMHR, FSHR, LHR, CYP19A1) factors was determined by quantitative RT-PCR using ROCHE LightCycler 480. Results No significant difference in GC or FF mRNA expression of pro- and anti-apoptotic factors could be demonstrated between IVF patients with (9 patients) or without (22 patients) clinical pregnancy. Each transcript investigated was detected in FF, but their levels were markedly reduced and independent of those in GCs. The number of retrieved oocytes was positively associated with GC AMHR (r=0.393, p=0.029), but the day of embryo transfer was negatively associated with GC LHR (r=-0.414, p=0.020) and GC FSHR transcripts (r=-0.535, p=0.002). When pregnancy positive group was analysed separately the impact of apoptosis- related gene expressions on some selected measures of IVF success could be observed. Strong positive relationship was found between gene expression levels of pro- and anti-apoptotic factors in GCs.ConclusionOur study provides only marginal evidences for the apoptosis dependence of IVF outcome and suggests that the apoptosis process induces adaptive increases of the anti-apoptotic gene expression to attenuate apoptosis and to protect cell survival.

scholarly journals Salmonellaexpresses foreign genes during infection by degrading their silencer

2020 ◽  
Vol 117 (14) ◽  
pp. 8074-8082 ◽  
Author(s):  
Jeongjoon Choi ◽  
Eduardo A. Groisman
Keyword(s):  
Binding Sites ◽  
Bacterial Species ◽  
Foreign Gene ◽  
Regulatory Sequences ◽  
Intracellular Pathogen ◽  
Heat Stable ◽  
Serovar Typhimurium ◽  
Foreign Dna ◽  
Foreign Genes ◽  
Virulence Regulator

The heat-stable nucleoid structuring (H-NS, also referred to as histone-like nucleoid structuring) protein silences transcription of foreign genes in a variety of Gram-negative bacterial species. To take advantage of the products encoded in foreign genes, bacteria must overcome the silencing effects of H-NS. Because H-NS amounts are believed to remain constant, overcoming gene silencing has largely been ascribed to proteins that outcompete H-NS for binding to AT-rich foreign DNA. However, we report here that the facultative intracellular pathogenSalmonella entericaserovar Typhimurium decreases H-NS amounts 16-fold when inside macrophages. This decrease requires both the protease Lon and the DNA-binding virulence regulator PhoP. The decrease in H-NS abundance reduces H-NS binding to foreign DNA, allowing transcription of foreign genes, including those required for intramacrophage survival. The purified Lon protease degraded free H-NS but not DNA-bound H-NS. By displacing H-NS from DNA, the PhoP protein promoted H-NS proteolysis, thereby de-repressing foreign genes—even those whose regulatory sequences are not bound by PhoP. The uncovered mechanism enables a pathogen to express foreign virulence genes during infection without the need to evolve binding sites for antisilencing proteins at each foreign gene.

scholarly journals Differential Gene Expression from Two Transcriptional Units in the cag Pathogenicity Island ofHelicobacter pylori

2001 ◽  
Vol 69 (7) ◽  
pp. 4202-4209 ◽  
Author(s):  
Elizabeth A. Joyce ◽  
Joanne V. Gilbert ◽  
Kathryn A. Eaton ◽  
Andrew Plaut ◽  
Andrew Wright
Keyword(s):  
Gene Expression ◽  
Cell Monolayer ◽  
Regulatory Region ◽  
Immunological Response ◽  
Pathogenicity Island ◽  
Gastric Disease ◽  
Regulatory Sequences ◽  
H Pylori

ABSTRACT Infection with Helicobacter pylori strains containing the cag Pathogenicity Island (cag PAI) is strongly correlated with the development of severe gastric disease, including gastric and duodenal ulceration, mucosa-associated lymphoid tissue lymphoma, and gastric carcinoma. Although in vitro studies have demonstrated that the expression of genes within the cag PAI leads to the activation of a strong host inflammatory response, the functions of mostcag gene products and how they work in concert to promote an immunological response are unknown. We developed a transcriptional reporter that utilizes urease activity and in which nine putative regulatory sequences from the cag PAI were fused to theH. pylori ureB gene. These fusions were introduced in single copies onto the H. pylori chromosome without disruption of the cag PAI. Our analysis indicated that while each regulatory region confers a reproducible amount of promoter activity under laboratory conditions, they differ widely in levels of expression. Transcription initiating upstream of cag15 and upstream of cag21 is induced when the respective fusion strains are cocultured with an epithelial cell monolayer. Results of mouse colonization experiments with an H. pylori strain carrying the cag15-ureB fusion suggested that this putative regulatory region appears to be induced in vivo, demonstrating the importance of the urease reporter as a significant development toward identifying in vivo-induced gene expression in H. pylori.

scholarly journals Expression Pattern of the Alpha-Kafirin Promoter Coupled with a Signal Peptide fromSorghum bicolorL. Moench

2012 ◽  
Vol 2012 ◽  
pp. 1-8 ◽  
Author(s):  
Norazlina Ahmad ◽  
Rajnesh Sant ◽  
Milovan Bokan ◽  
Kathryn J. Steadman ◽  
Ian D. Godwin
Keyword(s):  
Gene Expression ◽  
Signal Peptide ◽  
Sweet Corn ◽  
Signal Sequence ◽  
Seed Storage ◽  
Foreign Gene ◽  
Regulatory Sequences ◽  
Fluorescent Properties ◽  
Gfp Expression ◽  
Foreign Gene Expression

Regulatory sequences with endosperm specificity are essential for foreign gene expression in the desired tissue for both grain quality improvement and molecular pharming. In this study, promoters of seed storage α-kafirin genes coupled with signal sequence (ss) were isolated fromSorghum bicolorL. Moench genomic DNA by PCR. The α-kafirin promoter (α-kaf) contains endosperm specificity-determining motifs, prolamin-box, the O2-box 1, CATC, and TATA boxes required for α-kafirin gene expression in sorghum seeds. The constructs pMB-Ubi-gfpand pMB-kaf-gfpwere microprojectile bombarded into various sorghum and sweet corn explants. GFP expression was detected on all explants using the Ubi promoter but only in seeds for the α-kaf promoter. This shows that the α-kaf promoter isolated was functional and demonstrated seed-specific GFP expression. The constructs pMB-Ubi-ss-gfpand pMB-kaf-ss-gfpwere also bombarded into the same explants. Detection of GFP expression showed that the signal peptide (SP)::GFP fusion can assemble and fold properly, preserving the fluorescent properties of GFP.

Effects of the HDAC inhibitor scriptaid on the in vitro development of bovine embryos and on imprinting gene expression levels

2018 ◽  
Vol 110 ◽  
pp. 79-85 ◽  
Author(s):  
R. Laguna-Barraza ◽  
M.J. Sánchez-Calabuig ◽  
A. Gutiérrez-Adán ◽  
D. Rizos ◽  
S. Pérez-Cerezales
Keyword(s):  
Gene Expression ◽  
Hdac Inhibitor ◽  
Bovine Embryos ◽  
In Vitro Development ◽  
Expression Levels ◽  
Imprinting Gene ◽  
Vitro Development ◽  
Gene Expression Levels

5 PROPYLENE GLYCOL FEEDING SUPPLEMENTATION MODIFIES INSULIN-LIKE GROWTH FACTOR SYSTEM GENE EXPRESSION IN CUMULUS–OOCYTE COMPLEXES AND THE EXPRESSION OF SELECTED CANDIDATE GENES IN EMBRYOS PRODUCED IN VITRO IN FEED-RESTRICTED HEIFERS

2015 ◽  
Vol 27 (1) ◽  
pp. 95
Author(s):  
G. Gamarra ◽  
C. Ponsart ◽  
S. Lacaze ◽  
F. Nuttinck ◽  
P. Mermillod ◽  
...  
Keyword(s):  
Gene Expression ◽  
Growth Rate ◽  
Growth Factor ◽  
Propylene Glycol ◽  
Dietary Supplementation ◽  
Cumulus Cells ◽  
Oestrous Cycle ◽  
Expression Levels ◽  
Igf System

Dietary supplementation with propylene glycol (PG) increases the rate of grade 1 embryos produced from feed restricted females (Gamarra et al. 2014 Reprod. Fertil. Dev.). The aim of this study was to evaluate if a PG feeding supplement could modify the expression profile of selected candidate genes that are important for in vitro embryo development and the gene expression patterns of the insulin-like growth factor (IGF) system in oocytes and cumulus cells in feed-restricted heifers. Feed-restricted heifers (n = 16, growth rate of 600 g day–1) received a single daily drench of 400 mL of water (group restricted, R) from Day 1 to Day 9 of a first synchronized oestrous cycle followed by 400 mL of PG from Day 1 to Day 9 of the second synchronized oestrous cycle (group restricted + PG, RPG). Ovum pick-up (OPU) was performed following superovulation, on Day 5 of the oestrous cycle to produce embryos in vitro and on Day 9 without superovulation to obtain oocytes and cumulus cells. The same protocol was used in control animals (n = 6, growth rate of 800 g day–1). Real-time PCR was used to determine the relative abundance of genes involved in lipid metabolism and storage (PLIN2, SCD), energy metabolism (ATP5A1, GLUT1), membrane permeability (AQP3), epigenetic marks (DNMT3a), apoptosis (BAX, TP53), and protein processing (HSPA9B) in grade 1 blastocysts, IGF1, IGF1R, IGFBP2, IGFBP4 in cumulus cells, and IGF1R and IGFBP2 in oocytes. Mann-Whitney nonparametric tests were performed to analyse gene expression results. The expression of PLIN2, ATP5A1, GLUT1, AQP3, DNMT3a, BAX, and HSPA9B were decreased in embryos collected from restricted compared with control animals. The expression levels of these genes were restored when females were supplemented with PG. The expression of TP53 and SCD were not affected. In cumulus cells, the expression levels of IGF1, IGF1R, and IGFBP4 were decreased in restricted compared with control animals. The expression levels of IGF1 and IGF1R were restored with PG supplementation. No differences were observed for the IGFBP2 gene. In the oocytes, no differences were observed for the expression levels of IGF1R and IGFBP2 genes. In conclusion, this work shows for the first time that feed restriction and dietary supplementation by PG in heifers produced changes in gene expression in blastocysts and modified the pattern of the IGF system in cumulus cells. These results suggest the existence of an epigenetic regulation induced by PG during follicular growth, which can regulate the level of gene expression up to the blastocyst stage. In general, PG supplementation of feed-restricted donors restored gene expression at the levels observed after normal feeding.

cMET expression in HER2+ MBC patients with first-line lapatinib (L) treatment

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. 1073-1073
Author(s):  
Y. Liu ◽  
L. Liu ◽  
H. Shi ◽  
J. G. Greger ◽  
K. D. Jackson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Los Angeles ◽  
Clinical Outcome ◽  
Progression Free Survival ◽  
First Line ◽  
Free Survival ◽  
Expression Levels ◽  
Combination Study ◽  
Her 2

1073 Background: Overexpression of MET correlates with poor prognosis in breast cancer (Garcia et al., 2007) and is a factor associated with decreased sensitivity to L in HER2+ breast tumor cell lines in vitro (Liu et al., submitted). To test whether MET expression was associated with resistance to L in the clinic we evaluated baseline tumor MET expression levels and clinical outcome to L in 64 patients who participated in the EGF20009 trial of monotherapy L as first-line treatment in HER2+ advanced or MBC. Methods: RNA was extracted from FFPE tumors and MET and HER-2 gene expression was measured by qRT-PCR (Response Genetics, Inc., Los Angeles, CA). The correlation between expression levels of MET, HER2, and clinical outcome (overall response and progression free survival) was performed using JMP software. Results: A trend towards an association with increased MET expression and decreased response (p < 0.054) was observed.. Patients with high HER2 and low MET gene expression had the longest PFS (median difference = ∼9 weeks) compared to patients with low HER2 and high MET gene expression (p < 0.0038). Conclusions: These data support investigating a combination study of L and GSK1363089, a multi-kinase MET inhibitor, in HER2+ BC patients with high MET gene expression. [Table: see text]

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