scholarly journals Preparation of North American Type II PRRSV Infectious Clone Expressing Green Fluorescent Protein

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Liyue Wang ◽  
Kao Zhang ◽  
Hongyu Lin ◽  
Wenyan Li ◽  
Jiexia Wen ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
North American ◽  
Fluorescent Protein ◽  
Infectious Clone ◽  
Type Ii ◽  
Nsp2 Gene ◽  
Gfp Gene ◽  
Overlap Pcr ◽  
Green Fluorescent ◽  
North American Type

Porcine reproductive and respiratory syndrome virus (PRRSV) is still one of the most important infectious diseases threatening the swine industry. To construct North American type II PRRSV infectious clone containing green fluorescent protein (GFP) gene, we amplify gfp gene, flanked by PRRSV Nsp2 gene fragments upstream and downstream, using overlap PCR method from pcDNA-EF1-GFP plasmid and FL12 plasmid containing PRRSV infectious genome as the templates. The Nsp2 fragment-flanked gfp gene was inserted into Nsp2 gene of the FL12 plasmid bySpeI andXhoI sites to generate PRRSV infectious recombinant plasmid (FL12-GFP) containing gfp gene. The recombinant PRRSV expressing GFP (PRRSV-GFP) was rescued in baby hamster kidney-21 (BHK-21) cells by transfecting PRRSV mRNA synthesizedin vitroand amplified in Marc-145 cells. The PRRSV-GFP infectivity and replication capacity were identified. Results showed that, by adopting overlap PCR strategy, the gfp gene was successfully inserted into and fused with PRRSV Nsp2 gene in the PRRSV infectious clone plasmid FL-12 to generate FL12-GFP plasmid. The recombinant PRRSV-GFP was generated through transfecting PRRSV mRNA in BHK-2 cells. Like its parental virus, the recombinant PRRSV-GFP maintains its infectivity to Marc-145 cells and porcine alveolar macrophages (PAMs). This study provides essential conditions for further investigation on PRRSV.

scholarly journals A Full-Length cDNA Infectious Clone of North American Type 1 Porcine Reproductive and Respiratory Syndrome Virus: Expression of Green Fluorescent Protein in the Nsp2 Region

2006 ◽  
Vol 80 (23) ◽  
pp. 11447-11455 ◽  
Author(s):  
Ying Fang ◽  
Raymond R. R. Rowland ◽  
Michael Roof ◽  
Joan K. Lunney ◽  
Jane Christopher-Hennings ◽  
...  
Keyword(s):  
United States ◽  
Green Fluorescent Protein ◽  
North American ◽  
Fluorescent Protein ◽  
Infectious Clone ◽  
The United States ◽  
Respiratory Syndrome Virus ◽  
Green Fluorescent ◽  
North American Type

ABSTRACT The recent emergence of a unique group of North American type 1 porcine reproductive and respiratory syndrome virus (PRRSV) in the United States presents new disease control problems for a swine industry that has already been impacted seriously by North American type 2 PRRSV. In this study, a full-length cDNA infectious clone was generated from a low-virulence North American type 1 PRRSV isolate, SD01-08. In vitro studies demonstrated that the cloned virus maintained growth properties similar to those of the parental virus. Virological, pathological, and immunological observations from animals challenged with cloned viruses were similar to those from animals challenged with the parental virus and a modified live virus vaccine. To further explore the potential use as a viral backbone for expressing foreign genes, the green fluorescent protein (GFP) was inserted into a unique deletion site located at amino acid positions 348 and 349 of the predicted Nsp2 region in the virus, and expression of the Nsp2-GFP fusion protein was visualized by fluorescent microscopy. The availability of this North American type 1 infectious clone provides an important research tool for further study of the basic viral biology and pathogenic mechanisms of this group of type 1 PRRSV in the United States.

scholarly journals Development and Characterization of a cDNA-Launch Recombinant Simian Hemorrhagic Fever Virus Expressing Enhanced Green Fluorescent Protein: ORF 2b’ Is Not Required for In Vitro Virus Replication

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 632
Author(s):  
Yingyun Cai ◽  
Shuiqing Yu ◽  
Ying Fang ◽  
Laura Bollinger ◽  
Yanhua Li ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Cell Lines ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Infectious Clone ◽  
Hemorrhagic Fever ◽  
Simian Hemorrhagic Fever Virus ◽  
Hemorrhagic Fever Virus ◽  
Green Fluorescent

Simian hemorrhagic fever virus (SHFV) causes acute, lethal disease in macaques. We developed a single-plasmid cDNA-launch infectious clone of SHFV (rSHFV) and modified the clone to rescue an enhanced green fluorescent protein-expressing rSHFV-eGFP that can be used for rapid and quantitative detection of infection. SHFV has a narrow cell tropism in vitro, with only the grivet MA-104 cell line and a few other grivet cell lines being susceptible to virion entry and permissive to infection. Using rSHFV-eGFP, we demonstrate that one cricetid rodent cell line and three ape cell lines also fully support SHFV replication, whereas 55 human cell lines, 11 bat cell lines, and three rodent cells do not. Interestingly, some human and other mammalian cell lines apparently resistant to SHFV infection are permissive after transfection with the rSHFV-eGFP cDNA-launch plasmid. To further demonstrate the investigative potential of the infectious clone system, we introduced stop codons into eight viral open reading frames (ORFs). This approach suggested that at least one ORF, ORF 2b’, is dispensable for SHFV in vitro replication. Our proof-of-principle experiments indicated that rSHFV-eGFP is a useful tool for illuminating the understudied molecular biology of SHFV.

scholarly journals Mapping the Rubella Virus Subgenomic Promoter

2002 ◽  
Vol 76 (7) ◽  
pp. 3189-3201 ◽  
Author(s):  
Wen-Pin Tzeng ◽  
Teryl K. Frey
Keyword(s):  
Rubella Virus ◽  
Fluorescent Protein ◽  
Nonstructural Protein ◽  
Infectious Clone ◽  
Start Site ◽  
Structural Protein ◽  
Reading Frame ◽  
Gfp Gene ◽  
Green Fluorescent ◽  
Positive Strand Rna

ABSTRACT Rubella virus (RUB), the sole member of the Rubivirus genus in the Togaviridae family of positive-strand RNA viruses, synthesizes a single subgenomic (SG) RNA containing sequences from the 3′ end of the genomic RNA including the open reading frame (ORF) that encodes the virion proteins. The synthesis of SG RNA is initiated internally on a negative-strand, genome-length template at a site known as the SG promoter (SGP). Mapping the RUB SGP was initiated by using an infectious cDNA vector, dsRobo402/GFP, in which the region containing the SGP was duplicated (K. V. Pugachev, W.-P. Tzeng, and T. K. Frey, J. Virol. 74:10811-10815, 2000). In dsRobo402/GFP, the 5′-proximal nonstructural protein ORF (NS-ORF) is followed by the first SGP (SGP-1), the green fluorescent protein (GFP) gene, the second SGP (SGP-2), and the structural protein ORF. The duplicated SGP, SGP-2, contained nucleotides (nt) −175 to +76 relative to the SG start site, including the 3′ 127 nt of the NS-ORF and 47 nt between the NS-ORF and the SG start site. 5′ Deletions of SGP-2 to nt −40 (9 nt beyond the 3′ end of the NS-ORF) resulted in a wild-type (wt) phenotype in terms of virus replication and RNA synthesis. Deletions beyond this point impaired viability; however, the analysis was complicated by homologous recombination between SGP-1 and SGP-2 that resulted in deletion of the GFP gene and resurrection of viable virus with one SGP. Since the NS-ORF region was not necessary for SGP activity, subsequent mapping was done by using both replicon vectors, RUBrep/GFP and RUBrep/CAT, in which the SP-ORF is replaced with the reporter GFP and chloramphenical acetyltransferase genes, respectively, and the wt infectious clone, Robo402. In the replicon vectors, 5′ deletions to nt −26 resulted in the synthesis of SG RNA. In the infectious clone, deletions through nt −28 gave rise to viable virus. A series of short internal deletions confirmed that the region between nt −28 and the SG start site was essential for viability and showed that the repeated UCA triplet at the 5′ end of SG RNA was also required. Thus, the minimal SGP maps from nt −26 through the SG start site and appears to extend to at least nt +6, although a larger region is required for the generation of virus with a wt phenotype. Interestingly, while the positioning of the RUB SGP immediately adjacent the SG start site is thus similar to that of members of the genus Alphavirus, the other genus in the Togaviridae family, it does not include a region of nucleotide sequence homology with the alphavirus SGP that is located between nt −48 and nt −23 with respect to the SG start site in the RUB genome.

scholarly journals Transfer of Green Fluorescent Protein (GFP) Gene to Betta splendens Embryos by Transfection and Electroporation Methods

Omni-Akuatika ◽  
2018 ◽  
Vol 14 (2) ◽  
Author(s):  
Eni Kusrini ◽  
Alimuddin Alimuddin ◽  
Erma Primanita Hayuningtyas ◽  
Syuhada Restu Danupratama
Keyword(s):  
Green Fluorescent Protein ◽  
Gene Transfer ◽  
Expression Vector ◽  
Fluorescent Protein ◽  
Betta Splendens ◽  
Foreign Gene ◽  
Gfp Expression ◽  
Dna Concentration ◽  
Gfp Gene ◽  
Green Fluorescent

Transfection and electroporation method shave a high possibility to apply towards transgenic production of small eggs size fish species.  This study aimed to examine the potential of transfection and electroporation methods to use for transferring a foreign gene into betta fish (Betta splendens) embryos using green fluorescent protein (GFP) gene as a model.  Fish were spawned naturally in the ratio of male: female was 1:1, then a total of 200 eggs were taken for each treatment.  Transfection was performed for 30 minutes (room temperature of about 25 °C) at two-cell stage of embryos using transfast reagent.  Transfection reaction consisted of 0.75 µL transfast reagent, 0.25 µL GFP expression vector (DNA concentration: 50 µg/µL) and 99 µL NaCl solution (concentration: 0,95%).  Electroporation was performed using 125 volt cm-1, 3 times pulse frequency at one second interval and pulse length of 7 micro seconds.  A volume of 800 µL GFP expression vector solution (DNA concentration: 50 µg/ µL) in PBS was used for electroporation.  The successful of foreign gene transfer was determined by PCR method with GFP specific primers.  The results showed that hatching rate of eggs in transfection treatment was 67.08%, while the electroporation was 72.09%.  Survival of larvae in transfection treatment was 73.00%, while the electroporation was 75.00%.  The results of PCR analysis showed that transfection method allowed 65% of the survived fish carrying GFP gene, whereas the electroporation method was 70%.  Thus, foreign gene transfer in betta fish can be conducted using the transfection and electroporation methods. 

scholarly journals Infection, Dissemination, and Transmission of a West Nile Virus Green Fluorescent Protein Infectious Clone byCulex pipiens quinquefasciatusMosquitoes

2010 ◽  
Vol 10 (3) ◽  
pp. 267-274 ◽  
Author(s):  
Charles E. McGee ◽  
Alexandr V. Shustov ◽  
Konstantin Tsetsarkin ◽  
Ilya V. Frolov ◽  
Peter W. Mason ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
West Nile Virus ◽  
Fluorescent Protein ◽  
Infectious Clone ◽  
West Nile ◽  
Green Fluorescent

scholarly journals A vector carrying the GFP gene (Green fluorescent protein) as a yeast marker for fermentation processes

2000 ◽  
Vol 57 (4) ◽  
pp. 713-716 ◽  
Author(s):  
Luiz Humberto Gomes ◽  
Keila Maria Roncato Duarte ◽  
Felipe Gabriel Andrino ◽  
Ana Maria Brancalion Giacomelli ◽  
Flavio Cesar Almeida Tavares
Keyword(s):  
Green Fluorescent Protein ◽  
Early Detection ◽  
Fluorescent Protein ◽  
Low Frequency ◽  
Yeast Cells ◽  
Gfp Gene ◽  
Spoilage Yeasts ◽  
Adh2 Promoter ◽  
Pure Cultures ◽  
Green Fluorescent

Contaminant yeasts spoil pure culture fermentations and cause great losses in quality and product yields. They can be detected by a variety of methods although none being so efficient for early detection of contaminant yeast cells that appear at low frequency. Pure cultures bearing genetic markers can ease the direct identification of cells and colonies among contaminants. Fast and easy detection are desired and morphological markers would even help the direct visualization of marked pure cultures among contaminants. The GFP gene for green fluorescent protein of Aquorea victoria, proved to be a very efficient marker to visualize transformed cells in mixed populations and tissues. To test this marker in the study of contaminated yeast fermentations, the GFP gene was used to construct a vector under the control of the ADH2 promoter (pYGFP3). Since ADH2 is repressed by glucose the expression of the protein would not interfere in the course of fermentation. The transformed yeasts with the vector pYGFP3 showed high stability and high bioluminescence to permit identification of marked cells among a mixed population of cells. The vector opens the possibility to conduct further studies aiming to develop an efficient method for early detection of spoilage yeasts in industrial fermentative processes.

Sign in / Sign up

Export Citation Format

Share Document