Double-Stranded RNA-Degrading Enzymes Reduce the Efficiency of RNA Interference in Plutella xylostella

DsRNA-degrading enzymes (dsRNases) have been recognized as important factors in reducing RNA interference (RNAi) efficiency in different insect species. However, dsRNases in Plutella xylostella are still unknown. We identified the full-length cDNAs of PxdsRNase1, PxdsRNase2, PxdsRNase3, and PxdsRNase4. Gene expression profile showed that PxdsRNase1 was mainly expressed in the hemolymph; and that PxdsRNase2 and PxdsRNase3 were mainly expressed in the intestinal tract. The expression of PxCht (Chitinase of P. xylostella) in P. xylostella larvae injected with the mixture of dsPxCht (dsRNA of PxCht) and dsPxdsRNase1 (dsRNA of PxdsRNase1), dsPxdsRNase2 (dsRNA of PxdsRNase2), or dsPxdsRNase3 (dsRNA of PxdsRNase3) was significantly higher than that in the larvae injected with the mixture of dsGFP (dsRNA of green fluorescent protein gene, GFP) and dsPxCht; the transcription level of PxCht in the larvae feeding on the mixture of dsPxCht and dsPxdsRNase1, dsPxdsRNase2, or dsPxdsRNase3 was significantly higher than that in the larvae feeding on the mixture of dsPxCht and dsGFP. The recombinant protein of PxdsRNase1 degraded dsRNA rapidly, PxdsRNase3 cleaved dsRNA without complete degradation, and PxdsRNase2 could not degrade dsRNA in vitro. These results suggested that PxdsRNases1, PxdsRNases2, and PxdsRNases3 were involved in the dsRNA degradation to reduce RNAi efficiency with different mechanisms.

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Non-Target Effects of Green Fluorescent Protein (GFP)-Derived Double-Stranded RNA (dsRNA-GFP) Used in Honey Bee RNA Interference (RNAi) Assays

Insects ◽

10.3390/insects4010090 ◽

2013 ◽

Vol 4 (1) ◽

pp. 90-103 ◽

Cited By ~ 43

Author(s):

Francis Nunes ◽

Aline Aleixo ◽

Angel Barchuk ◽

Ana Bomtorin ◽

Christina Grozinger ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Rna Interference ◽

Honey Bee ◽

Fluorescent Protein ◽

Double Stranded Rna ◽

Green Fluorescent ◽

Target Effects

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In Vitro Replication of Hepatitis E Virus (HEV) Genomes and of an HEV Replicon Expressing Green Fluorescent Protein

Journal of Virology ◽

10.1128/jvi.78.9.4838-4846.2004 ◽

2004 ◽

Vol 78 (9) ◽

pp. 4838-4846 ◽

Cited By ~ 125

Author(s):

Suzanne U. Emerson ◽

Hanh Nguyen ◽

Judith Graff ◽

David A. Stephany ◽

Alicia Brockington ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Cell Cultures ◽

Hepatitis E Virus ◽

Fluorescent Protein ◽

Hepatitis E ◽

Green Fluorescent Protein Gene ◽

Primate Cell ◽

Green Fluorescent ◽

Full Length Genome

ABSTRACT Hepatitis E virus (HEV) RNA replication occurred in seven of nine primate cell cultures transfected with in vitro transcripts of an infectious cDNA clone. Cell-to-cell spread did not occur in cell cultures, but rhesus monkeys inoculated with lysates of HEV-transfected PLC/PRF/5 and Huh-7 cells became infected with HEV. A replicon with the ORF2 and ORF3 genes deleted and replaced with the green fluorescent protein gene also replicated in the same primate cells that supported the replication of the full-length genome. Fluorescence-activated cell sorter analysis confirmed that the 7mG cap structure was critical for efficient infectivity, although replication could be initiated at a very low level in its absence. HEV virions were also able to infect a limited number of cells of certain lines.

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Virulence Gene Identification by Differential Fluorescence Induction Analysis of Staphylococcus aureus Gene Expression during Infection-Simulating Culture

Infection and Immunity ◽

10.1128/iai.70.3.1326-1333.2002 ◽

2002 ◽

Vol 70 (3) ◽

pp. 1326-1333 ◽

Cited By ~ 24

Author(s):

William P. Schneider ◽

Sun K. Ho ◽

Jillian Christine ◽

Monique Yao ◽

Andrea Marra ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Fluorescent Protein ◽

Virulence Gene ◽

Bioinformatic Analysis ◽

Growth Conditions ◽

Fluorescence Induction ◽

Green Fluorescent Protein Gene ◽

Infection Model ◽

Green Fluorescent

ABSTRACT We have employed a strategy utilizing differential fluorescence induction (DFI) in an effort to identify Staphylococcus aureus genes whose products can be targeted for antimicrobial drug development. DFI allows identification of promoters preferentially active under given growth conditions on the basis of their ability to drive expression of a promoterless green fluorescent protein gene (gfp). A plasmid-based promoter trap library was constructed of 200- to 1,000-bp fragments of S. aureus genomic DNA fused to gfp, and clones with active promoters were isolated under seven different in vitro growth conditions simulating infection. Six thousand two hundred sixty-seven clones with active promoters were screened to identify those that exhibited differential promoter activity. Bioinformatic analysis allowed the identification of 42 unique operons, containing a total of 61 genes, immediately downstream of the differentially active putative promoters. Replacement mutations were generated for most of these operons, and the abilities of the resulting mutants to cause infection were assessed in two different murine infection models. Approximately 40% of the mutants were attenuated in at least one infection model.

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Diverging Affinity of Tospovirus RNA Silencing Suppressor Proteins, NSs, for Various RNA Duplex Molecules

Journal of Virology ◽

10.1128/jvi.00595-10 ◽

2010 ◽

Vol 84 (21) ◽

pp. 11542-11554 ◽

Cited By ~ 73

Author(s):

Esther Schnettler ◽

Hans Hemmes ◽

Rik Huismann ◽

Rob Goldbach ◽

Marcel Prins ◽

...

Keyword(s):

Rna Silencing ◽

Fluorescent Protein ◽

Micro Rna ◽

Impatiens Necrotic Spot Virus ◽

Small Interfering Rnas ◽

Double Stranded Rna ◽

Cell Extracts ◽

Green Fluorescent

ABSTRACT The tospovirus NSs protein was previously shown to suppress the antiviral RNA silencing mechanism in plants. Here the biochemical analysis of NSs proteins from different tospoviruses, using purified NSs or NSs containing cell extracts, is described. The results showed that all tospoviral NSs proteins analyzed exhibited affinity to small double-stranded RNA molecules, i.e., small interfering RNAs (siRNAs) and micro-RNA (miRNA)/miRNA* duplexes. Interestingly, the NSs proteins from tomato spotted wilt virus (TSWV), impatiens necrotic spot virus (INSV), and groundnut ringspot virus (GRSV) also showed affinity to long double-stranded RNA (dsRNA), whereas tomato yellow ring virus (TYRV) NSs did not. The TSWV NSs protein was shown to be capable of inhibiting Dicer-mediated cleavage of long dsRNA in vitro. In addition, it suppressed the accumulation of green fluorescent protein (GFP)-specific siRNAs during coinfiltration with an inverted-repeat-GFP RNA construct in Nicotiana benthamiana. In vivo interference of TSWV NSs in the miRNA pathway was shown by suppression of an enhanced GFP (eGFP) miRNA sensor construct. The ability to stabilize miRNA/miRNA* by different tospovirus NSs proteins in vivo was demonstrated by increased accumulation and detection of both miRNA171c and miRNA171c* in tospovirus-infected N. benthamiana. All together, these data suggest that tospoviruses interfere in the RNA silencing pathway by sequestering siRNA and miRNA/miRNA* molecules before they are uploaded into their respective RNA-induced silencing complexes. The observed affinity to long dsRNA for only a subset of the tospoviruses studied is discussed in light of evolutional divergence and their ancestral relation to the animal-infecting members of the Bunyaviridae.

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Single-Copy Green Fluorescent Protein Gene Fusions Allow Accurate Measurement of Salmonella Gene Expression In Vitro and during Infection of Mammalian Cells

Applied and Environmental Microbiology ◽

10.1128/aem.69.12.7480-7491.2003 ◽

2003 ◽

Vol 69 (12) ◽

pp. 7480-7491 ◽

Cited By ~ 172

Author(s):

Isabelle Hautefort ◽

Maria José Proença ◽

Jay C. D. Hinton

Keyword(s):

Gene Expression ◽

Green Fluorescent Protein ◽

Mammalian Cells ◽

Fluorescent Protein ◽

Single Copy ◽

Green Fluorescent Protein Gene ◽

Gene Fusions ◽

Bacterial Gene ◽

Green Fluorescent

ABSTRACT We developed a reliable and flexible green fluorescent protein (GFP)-based system for measuring gene expression in individual bacterial cells. Until now, most systems have relied upon plasmid-borne gfp gene fusions, risking problems associated with plasmid instability. We show that a recently developed GFP variant, GFP+, is suitable for assessing bacterial gene expression. Various gfp+ transcriptional fusions were constructed and integrated as single copies into the chromosome of Salmonella enterica serovar Typhimurium. A comparison of the expression levels of proU-lacZ and proU-gfp+ fusions showed that GFP+ reported proU activity in individual Salmonella cells as accurately as β-galactosidase reported activity for entire populations. The single-copy gfp+ fusions were ideal for monitoring up- and downregulation of Salmonella virulence genes. We discovered that in vitro induction of the SPI1gene prgH occurs only in a portion of the population and that the proportion varies with the growth phase. We determined the level of expression of the SPI2 gene ssaG in bacteria released from murine macrophages. Our results demonstrate for the first time that single-copy GFP+ fusions reliably report gene expression in simple and complex environments. This approach promises to allow accurate measurement of gene expression in individual bacteria during animal infection.

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307 TRANSGENIC OVINE EMBRYOS BY ARTIFICIAL INSEMINATION, IN VITRO FERTILIZATION AND INTRACYTOPLASMIC SPERM INJECTION

Reproduction Fertility and Development ◽

10.1071/rdv21n1ab307 ◽

2009 ◽

Vol 21 (1) ◽

pp. 250 ◽

Cited By ~ 1

Author(s):

F. Pereyra-Bonnet ◽

A. Gibbons ◽

M. Cueto ◽

R. Fernández-Martín ◽

D. Salamone

Keyword(s):

Fluorescent Protein ◽

Blastocyst Stage ◽

Green Fluorescent Protein Gene ◽

Sperm Cells ◽

Normal Female ◽

Embryo Viability ◽

Vitro Fertilization ◽

Transgenic Embryos ◽

Green Fluorescent

Nowadays, transgenesis in animals constitutes an important tool for pharmacological protein production and livestock improvement. In 1971 Brackett first described that heterologous DNA can be introduced into a mammalian oocyte using sperm cells as vectors. We evaluated the capacity of AI, IVF and ICSI to produce transgenic embryos, in ovine, using sperm that had been exposed to a pCX-EGFP plasmid in Long and Short incubation protocols. The pCX-EGFP plasmid contains an enhanced green fluorescent protein gene (egfp) under the chimeric cytomegalovirus-IE-chicken β-actin enhancer-promoter control. Sperm/pCX-EGFP incubation was carried out by Long Incubation (2 h at 17°C in 200 μL of SFM medium: 100 mL contains glucose 1.2 g, Na citrate 1.0 g, EDTA 0.4 g, Citric acid 0.3 g, Trizma 0.6 g) and Short Incubation (5 min at 0–5°C in 10–100 μL of extender medium: 100 mL contains Na Citrate 2.8 g and EDTA 4 mg). For AI, Merino sheep (n = 17) were superovulated and inseminated with fresh semen (200 millions sperm/sheep) from eight Merino rams. The embryos were recovered by flushing the uterine horns by standard procedures. In IVF and ICSI, slaughterhouse oocytes were fertilized with frozen/thawed sperm. IVF was carried out in Brackett-Oliphant medium with 5 mm of caffeine, 20 IU mL–1 of heparin with 20 million sperm mL–1 during 5 h in an atmosphere of 5% CO2 in air. In ICSI, the spermatozoon was immobilized by breaking its tail and injected into MII oocytes. Immediately the oocytes were activated by incubation in TALP-HEPES with 5 μm ionomycin for 4 min, cultured in TCM199 for 3 h and transferred to a droplet of 1.9 mm DMAP for 3 h. Maturation and cultivation conditions were determined by standard operating procedures. All embryos were exposed to blue light (488 nm) to determine the percentage of morulae/blastocysts showing green fluorescence. Results are shown in Table 1. Statistical analysis was done by Fisher test. AI and IVF were able to produce a high percentage of morula and blastocyst stage, but were unable to produce transgenic embryos. In contrast, regardless of the sperm/plasmid incubation protocol, high percentages of transgenic morulae and blastocysts were always obtained by ICSI and the highest rate was achieved with Short Incubation (P < 0.05). In order to demonstrate ICSI-Short incubation embryo viability, two-day-old non-selected fluorescence embryos (n = 45), were transferred into the oviducts of five surrogate mothers. Pregnancy was diagnosed at day 25 (2/5; 40%), and one normal female lamb was recently born (1/5; 20%). In conclusion, our results show that in ovine, ICSI seems to be the only method for producing transgenic embryos using sperm cells as vectors. In addition the offspring born confirm the viability of these embryos. Table 1.Development and fluorescence expression of ovine embryos

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Engineering of Recombinant Sheep Pox Viruses Expressing Foreign Antigens

Microorganisms ◽

10.3390/microorganisms9051005 ◽

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.

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Development of a Fluorescent Tool for Studying Legionella bozemanae Intracellular Infection

Microorganisms ◽

10.3390/microorganisms9020379 ◽

2021 ◽

Vol 9 (2) ◽

pp. 379

Author(s):

Breanne M. Head ◽

Christopher I. Graham ◽

Teassa MacMartin ◽

Yoav Keynan ◽

Ann Karen C. Brassinga

Keyword(s):

Growth Kinetics ◽

Legionella Pneumophila ◽

Fluorescent Protein ◽

Disease Incidence ◽

Acanthamoeba Castellanii ◽

Infection Model ◽

Intracellular Infection ◽

Green Fluorescent

Legionnaires’ disease incidence is on the rise, with the majority of cases attributed to the intracellular pathogen, Legionella pneumophila. Nominally a parasite of protozoa, L. pneumophila can also infect alveolar macrophages when bacteria-laden aerosols enter the lungs of immunocompromised individuals. L. pneumophila pathogenesis has been well characterized; however, little is known about the >25 different Legionella spp. that can cause disease in humans. Here, we report for the first time a study demonstrating the intracellular infection of an L. bozemanae clinical isolate using approaches previously established for L. pneumophila investigations. Specifically, we report on the modification and use of a green fluorescent protein (GFP)-expressing plasmid as a tool to monitor the L. bozemanae presence in the Acanthamoeba castellanii protozoan infection model. As comparative controls, L. pneumophila strains were also transformed with the GFP-expressing plasmid. In vitro and in vivo growth kinetics of the Legionella parental and GFP-expressing strains were conducted followed by confocal microscopy. Results suggest that the metabolic burden imposed by GFP expression did not impact cell viability, as growth kinetics were similar between the GFP-expressing Legionella spp. and their parental strains. This study demonstrates that the use of a GFP-expressing plasmid can serve as a viable approach for investigating Legionella non-pneumophila spp. in real time.

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CRISPR/Cas9-based generation of a recombinant double-reporter pseudorabies virus and its characterization in vitro and in vivo

Veterinary Research ◽

10.1186/s13567-021-00964-4 ◽

2021 ◽

Vol 52 (1) ◽

Author(s):

Peng-Fei Fu ◽

Xuan Cheng ◽

Bing-Qian Su ◽

Li-Fang Duan ◽

Cong-Rong Wang ◽

...

Keyword(s):

Pseudorabies Virus ◽

Fluorescent Protein ◽

Antiviral Agents ◽

Firefly Luciferase ◽

Inhibitory Effect ◽

Economic Losses ◽

Green Fluorescent ◽

Swine Herds

AbstractPseudorabies, caused by pseudorabies virus (PRV) variants, has broken out among commercial PRV vaccine-immunized swine herds and resulted in major economic losses to the pig industry in China since late 2011. However, the mechanism of virulence enhancement of variant PRV is currently unclear. Here, a recombinant PRV (rPRV HN1201-EGFP-Luc) with stable expression of enhanced green fluorescent protein (EGFP) and firefly luciferase as a double reporter virus was constructed on the basis of the PRV variant HN1201 through CRISPR/Cas9 gene-editing technology coupled with two sgRNAs. The biological characteristics of the recombinant virus and its lethality to mice were similar to those of the parental strain and displayed a stable viral titre and luciferase activity through 20 passages. Moreover, bioluminescence signals were detected in mice at 12 h after rPRV HN1201-EGFP-Luc infection. Using the double reporter PRV, we also found that 25-hydroxycholesterol had a significant inhibitory effect on PRV both in vivo and in vitro. These results suggested that the double reporter PRV based on PRV variant HN1201 should be an excellent tool for basic virology studies and evaluating antiviral agents.

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Paracetamol modulates biofilm formation in Staphylococcus aureus clonal complex 8 strains

Scientific Reports ◽

10.1038/s41598-021-84505-1 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Andi R. Sultan ◽

Kirby R. Lattwein ◽

Nicole A. Lemmens-den Toom ◽

Susan V. Snijders ◽

Klazina Kooiman ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Biofilm Formation ◽

Fluorescent Protein ◽

Clonal Complex ◽

Regulatory Pathways ◽

Immune Modulator ◽

Viability Assay ◽

Analgesic Agents ◽

Green Fluorescent

AbstractStaphylococcus aureus biofilms are a major problem in modern healthcare due to their resistance to immune system defenses and antibiotic treatments. Certain analgesic agents are able to modulate S. aureus biofilm formation, but currently no evidence exists if paracetamol, often combined with antibiotic treatment, also has this effect. Therefore, we aimed to investigate if paracetamol can modulate S. aureus biofilm formation. Considering that certain regulatory pathways for biofilm formation and virulence factor production by S. aureus are linked, we further investigated the effect of paracetamol on immune modulator production. The in vitro biofilm mass of 21 S. aureus strains from 9 genetic backgrounds was measured in the presence of paracetamol. Based on biofilm mass quantity, we further investigated paracetamol-induced biofilm alterations using a bacterial viability assay combined with N-Acetylglucosamine staining. Isothermal microcalorimetry was used to monitor the effect of paracetamol on bacterial metabolism within biofilms and green fluorescent protein (GFP) promoter fusion technology for transcription of staphylococcal complement inhibitor (SCIN). Clinically relevant concentrations of paracetamol enhanced biofilm formation particularly among strains belonging to clonal complex 8 (CC8), but had minimal effect on S. aureus planktonic growth. The increase of biofilm mass can be attributed to the marked increase of N-Acetylglucosamine containing components of the extracellular matrix, presumably polysaccharide intercellular adhesion. Biofilms of RN6390A (CC8) showed a significant increase in the immune modulator SCIN transcription during co-incubation with low concentrations of paracetamol. Our data indicate that paracetamol can enhance biofilm formation. The clinical relevance needs to be further investigated.

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