scholarly journals Calcineurin Localizes to the Hyphal Septum in Aspergillus fumigatus: Implications for Septum Formation and Conidiophore Development

2008 ◽  
Vol 7 (9) ◽  
pp. 1606-1610 ◽  
Author(s):  
Praveen Rao Juvvadi ◽  
Jarrod R. Fortwendel ◽  
Nadthanan Pinchai ◽  
B. Zachary Perfect ◽  
Joseph Heitman ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Aspergillus Fumigatus ◽  
Filamentous Fungus ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Dependent Manner ◽  
Septum Formation ◽  
Calcineurin A ◽  
Green Fluorescent

ABSTRACT A functional calcineurin A fusion to enhanced green fluorescent protein (EGFP), CnaA-EGFP, was expressed in the Aspergillus fumigatus ΔcnaA mutant. CnaA-EGFP localized in actively growing hyphal tips, at the septa, and at junctions between the vesicle and phialides in an actin-dependent manner. This is the first study to implicate calcineurin in septum formation and conidiophore development of a filamentous fungus.

20 Generation of myostatin knockout horse embryos using clustered regularly interspaced short palindromic repeats/CRISPR-associated gene 9 and somatic cell nuclear transfer

2019 ◽  
Vol 31 (1) ◽  
pp. 136 ◽  
Author(s):  
G. Vichera ◽  
D. Viale ◽  
R. Olivera ◽  
V. Arnold ◽  
A. Grundnig ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Enhanced Green Fluorescent Protein ◽  
New Technologies ◽  
Fluorescent Protein ◽  
Cell Clone ◽  
Mesenchymal Cells ◽  
Negative Regulator ◽  
Dependent Manner ◽  
Mstn Gene ◽  
Green Fluorescent

Historically, livestock improvement by selective breeding was the principal selection force in animal production and welfare, but the desired phenotype may involve more than 1 generation. Nowadays, new technologies such as CRISPR/Cas9 could overpass these limits and improve animal quality by insertion or modification of the desired genotype. In this work, we aim to knock out the myostatin (MSTN) gene, a negative regulator of muscle mass development, in equine cells and to generate equine cloned embryos with this modified genotype. To achieve this, 1×105 equine fibroblasts were nucleofected with 5, 2, or 1μg of the plasmid hspCas9-2A-Puro V2.0 that codified for the Cas 9 nuclease and a single guide RNA (gRNA). Two different gRNA (gRNA 1 and gRNA 2) complementary to the first exon of the MSTN gene were designed and evaluated. Cells were also nucleofected with the enhanced green fluorescent protein-N1 plasmid in order to determine the transfection efficiency, obtaining more than 90% of enhanced green fluorescent protein+ cells in the 3 conditions. Forty-eight hours after nucleofection, cells were treated with 2.5μg mL−1 of puromycin for 48h to isolate cells that incorporated the plasmid. After that, clonal culture was achieved by plating individual cells in 96-well plates. The clones were then expanded individually and genomic DNA was isolated from each one, genotyped for the MSTN exon 1 locus by PCR amplification, and Sanger sequenced. Both gRNA had mutational activity, with 96% efficiency (24/25 clones) for gRNA 1 and 55.5% mutation activity (10/18 clones) for gRNA2. We obtained different genotypes depending on the gRNA and the dose that was used-gRNA 1: 1μg=57% wt/wt, 14% wt/mutX, and 29% mutX/mutX; 2μg=33.3% wt/wt, 33.3% mutX/mutY, and 33.3% mutX/mutX; 5μg=40% wt/wt and 60% mutX/mutY; gRNA 2: 1μg=17% wt/wt, 17% wt/mutX, 50% mutX/mutY, and 17% mutX/mutX; 2μg=67% mutX/mutY and 34% mutX/mutX; 5μg=54% mutX/mutY and 46% mutX/mutX. Two of the gRNA2 mutated cell lines were used for embryo generation by NT, 1 wt/mutX line (gRNA2-1ug-C2, heterozygote clone) and 1 mutX/mutX line (gRNA2-5ug-C13, homozygote clone). Before that, to assess specificity, the first 2 highly ranked off-target sites of gRNA2 were checked by Sanger sequencing in the selected clones, not observing modifications in their sequences. In both cases, we could generate edited MSTN equine cloned blastocysts: 3/153 (2%), 3/155 (2%), 8/140 (6%), and 9/73 (12%) for gRNA2-1μg-C2, gRNA2-5μg-C13, control fibroblasts, and control mesenchymal cells, respectively. In conclusion, genome edition by CRISPR/Cas9 is an efficient method to edit the genome of horse fibroblasts in a dose-dependent manner with apparent high specificity. Moreover, equine embryos can be generated with these cells with lower blastocyst rates than control fibroblasts of the same cell line or mesenchymal cells, probably due to higher cell passages needed for cell clone isolation and expansion. To the best of our knowledge, this is the first report of genome edited horse embryos.

scholarly journals Enhanced Green Fluorescent Protein (GFP) fluorescence after polyelectrolyte caging

2006 ◽  
Vol 14 (21) ◽  
pp. 9815 ◽  
Author(s):  
Alberto Diaspro ◽  
Silke Krol ◽  
Barbara Campanini ◽  
Fabio Cannone ◽  
Giuseppe Chirico
Keyword(s):  
Green Fluorescent Protein ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Gfp Fluorescence ◽  
Green Fluorescent

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.

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