scholarly journals Comparison of CRISPR–Cas9 Tools for Transcriptional Repression and Gene Disruption in the BEVS

Viruses ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 1925
Author(s):  
Mark R. Bruder ◽  
Sadru-Dean Walji ◽  
Marc G. Aucoin
Keyword(s):  
Transcriptional Repression ◽  
Gene Disruption ◽  
Protein Gene ◽  
Fluorescent Protein ◽  
Green Fluorescent Protein Gene ◽  
Proof Of Concept ◽  
Knock Out ◽  
Targeted Gene Disruption ◽  
Green Fluorescent ◽  
Affect Gene Expression

The generation of knock-out viruses using recombineering of bacmids has greatly accelerated scrutiny of baculovirus genes for a variety of applications. However, the CRISPR–Cas9 system is a powerful tool that simplifies sequence-specific genome editing and effective transcriptional regulation of genes compared to traditional recombineering and RNAi approaches. Here, the effectiveness of the CRISPR–Cas9 system for gene disruption and transcriptional repression in the BEVS was compared. Cell lines constitutively expressing the cas9 or dcas9 gene were developed, and recombinant baculoviruses delivering the sgRNA were evaluated for disruption or repression of a reporter green fluorescent protein gene. Finally, endogenous AcMNPV genes were targeted for disruption or downregulation to affect gene expression and baculovirus replication. This study provides a proof-of-concept that CRISPR–Cas9 technology may be an effective tool for efficient scrutiny of baculovirus genes through targeted gene disruption and transcriptional repression.

scholarly journals Targeted gene disruption reveals a role for vacuolin B in the late endocytic pathway and exocytosis

1998 ◽  
Vol 111 (1) ◽  
pp. 61-70 ◽  
Author(s):  
N. Jenne ◽  
R. Rauchenberger ◽  
U. Hacker ◽  
T. Kast ◽  
M. Maniak
Keyword(s):  
Gene Disruption ◽  
Fluorescent Protein ◽  
Fluid Phase ◽  
Endocytic Pathway ◽  
Knock Out ◽  
Targeted Gene Disruption ◽  
Green Fluorescent ◽  
Cytoplasmic Side ◽  
Dependent Mechanism

Cells of Dictyostelium discoideum take up fluid by macropinocytosis. The contents of macropinosomes are acidified and digested by lysosomal enzymes. Thereafter, an endocytic marker progresses in an F-actin dependent mechanism from the acidic lysosomal phase to a neutral post-lysosomal phase. From the post-lysosomal compartment indigestible remnants are released by exocytosis. This compartment is characterised by two isoforms of vacuolin, A and B, which are encoded by different genes. Fusions of the vacuolin isoforms to the green fluorescent protein associate with the cytoplasmic side of post-lysosomal vacuoles in vivo. Vacuolin isoforms also localise to patches at the plasma membrane. Since vacuolins have no homologies to known proteins and do not contain domains of obvious function, we investigated their role by knocking out the genes separately. Although the sequences of vacuolins A and B are about 80% identical, only deletion of the vacuolin B gene results in a defect in the endocytic pathway; the vacuolin A knock-out appeared to be phenotypically normal. In vacuolin B- mutants endocytosis is normal, but the progression of fluid-phase marker from acidic to neutral pH is impaired. Furthermore, in the mutants post-lysosomal vacuoles are dramatically increased in size and accumulate endocytic marker, suggesting a role for vacuolin B in targeting the vacuole for exocytosis.

Efficient Knockout of Transplanted Green Fluorescent Protein Gene in Medaka Using TALENs

2014 ◽  
Vol 16 (6) ◽  
pp. 674-683 ◽  
Author(s):  
Chao Qiu ◽  
Bin Cheng ◽  
Yunsheng Zhang ◽  
Rong Huang ◽  
Lanjie Liao ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Protein Gene ◽  
Fluorescent Protein ◽  
Green Fluorescent Protein Gene ◽  
Green Fluorescent

Stress-survival responses of a carbon-starvedp-nitrophenol-mineralizingMoraxellastrain in river water

2005 ◽  
Vol 51 (3) ◽  
pp. 223-229 ◽  
Author(s):  
Michael Moore ◽  
Jack Trevors ◽  
Hung Lee ◽  
Kam Tin Leung
Keyword(s):  
Green Fluorescent Protein ◽  
River Water ◽  
Water Samples ◽  
Protein Gene ◽  
Fluorescent Protein ◽  
Carbon Starvation ◽  
Green Fluorescent Protein Gene ◽  
River Water Samples ◽  
Stress Survival ◽  
Green Fluorescent

The effect of carbon starvation on the stress-resistant responses of a p-nitrophenol-mineralizing Moraxella strain was examined in both buffer and river water samples. The Moraxella strain showed optimal stress-resistant responses in a minimal salt buffer when carbon-starved for 1–2 d. In the buffer system, the 1- and 2-day carbon-starved Moraxella cultures survived about 150-, 200-, and 100-fold better than the non-starved cultures when exposed to 43.5 °C, 2.7 mol/L NaCl, and 500 µmol/L H2O2for 4 h, respectively. A green fluorescent protein gene- (gfp) labelled derivative of the Moraxella strain was used to examine the stress-resistant responses of the bacterium in natural river water microcosms. The carbon-starved gfp-labelled Moraxella strain also showed stress-resistant responses against heat, osmotic, and oxidative stresses in the river water samples. Despite the stress-tolerant capability of the carbon-starved gfp-labelled Moraxella cells, they did not exhibit any survival advantage over their non-starved counterparts when inoculated into river water microcosms and incubated at 10 and 22 °C for 14 d.Key words: carbon starvation, stress-survival responses, Moraxella, p-nitrophenol, green fluorescent protein gene.

Expression of the green fluorescent protein gene in conifer tissues

1997 ◽  
Vol 16 (5) ◽  
pp. 267-271 ◽  
Author(s):  
Lining Tian ◽  
Armand Séguin ◽  
Pierre J. Charest
Keyword(s):  
Green Fluorescent Protein ◽  
Protein Gene ◽  
Fluorescent Protein ◽  
Green Fluorescent Protein Gene ◽  
Green Fluorescent
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