scholarly journals The Nucleoprotein Is Required for Efficient Coronavirus Genome Replication

2004 ◽  
Vol 78 (22) ◽  
pp. 12683-12688 ◽  
Author(s):  
Fernando Almazán ◽  
Carmen Galán ◽  
Luis Enjuanes
Keyword(s):  
Heterologous Protein ◽  
Cytopathic Effect ◽  
Human Cell Line ◽  
Genome Replication ◽  
Bacterial Artificial Chromosomes ◽  
Artificial Chromosomes ◽  
Restriction Sites ◽  
Unique Restriction ◽  
Transmissible Gastroenteritis ◽  
Transmissible Gastroenteritis Coronavirus

ABSTRACT The construction of a set of transmissible gastroenteritis coronavirus (TGEV)-derived replicons as bacterial artificial chromosomes is reported. These replicons were generated by sequential deletion of nonessential genes for virus replication, using a modified TGEV full-length cDNA clone containing unique restriction sites between each pair of consecutive genes. Efficient activity of TGEV replicons was associated with the presence of the nucleoprotein provided either in cis or in trans. TGEV replicons were functional in several cell lines, including the human cell line 293T, in which no or very low cytopathic effect was observed, and expressed high amounts of heterologous protein.

scholarly journals Cross-Species RNAi Rescue Platform in Drosophila melanogaster

Genetics ◽  
2009 ◽  
Vol 183 (3) ◽  
pp. 1165-1173 ◽  
Author(s):  
Shu Kondo ◽  
Matthew Booker ◽  
Norbert Perrimon
Keyword(s):  
Cell Culture ◽  
Drosophila Melanogaster ◽  
Large Scale ◽  
Transgenic Animals ◽  
Selection Marker ◽  
Bacterial Artificial Chromosomes ◽  
Loss Of Function ◽  
Artificial Chromosomes ◽  
Target Effects

RNAi-mediated gene knockdown in Drosophila melanogaster is a powerful method to analyze loss-of-function phenotypes both in cell culture and in vivo. However, it has also become clear that false positives caused by off-target effects are prevalent, requiring careful validation of RNAi-induced phenotypes. The most rigorous proof that an RNAi-induced phenotype is due to loss of its intended target is to rescue the phenotype by a transgene impervious to RNAi. For large-scale validations in the mouse and Caenorhabditis elegans, this has been accomplished by using bacterial artificial chromosomes (BACs) of related species. However, in Drosophila, this approach is not feasible because transformation of large BACs is inefficient. We have therefore developed a general RNAi rescue approach for Drosophila that employs Cre/loxP-mediated recombination to rapidly retrofit existing fosmid clones into rescue constructs. Retrofitted fosmid clones carry a selection marker and a phiC31 attB site, which facilitates the production of transgenic animals. Here, we describe our approach and demonstrate proof-of-principle experiments showing that D. pseudoobscura fosmids can successfully rescue RNAi-induced phenotypes in D. melanogaster, both in cell culture and in vivo. Altogether, the tools and method that we have developed provide a gold standard for validation of Drosophila RNAi experiments.

Homologous Recombination Using Bacterial Artificial Chromosomes

2015 ◽  
Vol 2015 (2) ◽  
pp. pdb.prot072397
Author(s):  
Cary Lai ◽  
Tobias Fischer ◽  
Elizabeth Munroe
Keyword(s):  
Homologous Recombination ◽  
Bacterial Artificial Chromosomes ◽  
Artificial Chromosomes

scholarly journals The effect of vitamin and mineral supplements on the replication of coronaviruses in the SPEV cell culture

2021 ◽  
Author(s):  
V.N. Laskavyy ◽  
T.I. Polyanina ◽  
V.V. Laskavaya ◽  
V.T. Nochevny
Keyword(s):  
Reference Strain ◽  
Cytopathic Effect ◽  
Virus Titer ◽  
Transmissible Gastroenteritis Virus ◽  
Infectious Dose ◽  
Mineral Supplements ◽  
Porcine Transmissible Gastroenteritis Virus ◽  
Order Of Magnitude ◽  
Transmissible Gastroenteritis ◽  
Strain Virus

The article describes studies on the effect of vitamin and mineral supplements (VMS) on the replication of coronavirus in tissue culture. The cytopathic effect of the porcine transmissible gastroenteritis virus (strain TO36SD192 (Japan) and the vaccine strain "RIMS" was studied on the SPEV cell line in the presence of mineral and vitamin supplements (VMS) with an increased content of vitamins A and D. The results of the study showed that the introduction of vitamin and mineral supplements into the growth medium provides an increase in the yield of the RIMS virus by 16.7 - 23%. Interestingly, at the minimum infectious dose, the presence or absence of VMS does not in any way affect the titer of the virus of the reference strain of the coronavirus TO36SD192. However, an increase in the infectious dose by only one order of magnitude (1000 TDC50 / ml) stimulates an increase in the virus titer by 22.5% in the presence of VMS. With the addition of VMS, the RIMS strain reproduced more actively and the virus titer in the experiment with the addition of VMS was 30% higher than in the control (without VMS). A further increase in the infectious dose showed a significant increase in the virus titer from 13.4% to 6.25%. It should be noted that the TO36SD192 virus, well adapted to the SPEV culture, can be compared with viruses that cause seasonal ARVI in humans, which are also well adapted in the human body, and the use of VMS during the peak of the epidemic can stimulate viral replication. Since the RIMS strain virus is poorly adapted to the SPEV culture, its action can be compared with a virus that has entered the human population, for example, SARS-CoV-2. This means that taking vitamins at any infectious dose (during an epidemic) will stimulate the disease.

scholarly journals Chromatin Structure of the Simian Virus 40 Late Promoter: a Deletional Analysis

1999 ◽  
Vol 73 (3) ◽  
pp. 1990-1997 ◽  
Author(s):  
Michael Friez ◽  
RaeJean Hermansen ◽  
Barry Milavetz
Keyword(s):  
Dna Sequences ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Late Promoter ◽  
Reporter System ◽  
Free Region ◽  
Restriction Sites ◽  
Unique Restriction ◽  
Minimal Sequence ◽  
Nuclease Sensitivity

ABSTRACT The goal of this study was to determine the minimal sequence within the simian virus 40 (SV40) late promoter region, nucleotides (nt) 255 to 424, capable of phasing nucleosomes as measured by its ability to confer the greatest endonuclease sensitivity on adjacent DNA sequences. To identify the minimal sequence, a deletional analysis of the late region was performed by utilizing a SV40 recombinant reporter system. The reporter system consisted of a series of unique restriction sites introduced into SV40 at nt 2666. The unique restriction sites allowed the insertion of test sequences as well as measurement of conferred endonuclease sensitivity. The results of the deletional analysis demonstrated that constructs capable of conferring the greatest nuclease sensitivities consistently included nt 255 to 280. The activator protein 4 (AP-4) and GTIIC transcription factor binding sequences lie within this region and were analyzed individually. Their abilities to confer nuclease sensitivity upon the reporter nearly matched that of the entire late domain. These results suggest that transcription factors AP-4 and transcription-enhancing factor which binds the GTIIC sequence are able to confer significant levels of nuclease sensitivity and are likely involved in the formation of the SV40 nucleosome-free region.

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