scholarly journals Expression of Secreted Neutrophil Gelatinase-Associated Lipocalin in 293T Cell Using the Inducible Dual-Function System

Processes ◽  
2021 ◽  
Vol 9 (5) ◽  
pp. 855
Author(s):  
Somphot Saoin ◽  
Chatikorn Boonkrai ◽  
Trairak Pisitkun ◽  
Chiraphat Kloypan ◽  
Sawitree Nangola
Keyword(s):  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Transfection Efficiency ◽  
Signal Sequence ◽  
Kidney Injury ◽  
293T Cell ◽  
Dual Function ◽  
Neutrophil Gelatinase Associated Lipocalin ◽  
Green Fluorescent ◽  
Neutrophil Gelatinase

Neutrophil gelatinase-associated lipocalin (NGAL) has emerged as a promising biomarker for the early prediction of acute kidney injury (AKI). The production of recombinant NGAL is considered to be necessary for the development of a detection method. This study intended to express the recombinant NGAL protein in 293T cell under the Tet-On inducible system and human serum albumin signal sequence (HSA-SS). The transfection efficiency and protein modulation were assessed by detecting the expression of the enhanced green fluorescent protein (EGFP) and secreted NGAL protein. Both proteins were detected only in the presence of a doxycycline (Dox) inducer. Cell toxicity was not found under any conditions. Moreover, a higher level of soluble NGAL protein in the supernatant secreted by HSA-SS compared with a native signal peptide (Nat-SS) was observed. In summary, this work successfully optimized the conditions for induction of NGAL expression. This system will provide as an efficient strategy to produce other recombinant proteins secreted from a mammalian cell.

A quantitative method for normalization of transfection efficiency using enhanced green fluorescent protein

2005 ◽  
Vol 342 (2) ◽  
pp. 341-344 ◽  
Author(s):  
Dineshkumar H. Dandekar ◽  
Manish Kumar ◽  
Jayashree S. Ladha ◽  
Krishna N. Ganesh ◽  
Debashis Mitra
Keyword(s):  
Green Fluorescent Protein ◽  
Quantitative Method ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Transfection Efficiency ◽  
Green Fluorescent

scholarly journals Generation of a Recombinant Akabane Virus Expressing Enhanced Green Fluorescent Protein

2015 ◽  
Vol 89 (18) ◽  
pp. 9477-9484 ◽  
Author(s):  
Akiko Takenaka-Uema ◽  
Yousuke Murata ◽  
Fumihiro Gen ◽  
Yukari Ishihara-Saeki ◽  
Ken-ichi Watanabe ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Signal Sequence ◽  
Rift Valley Fever Virus ◽  
Brain Slices ◽  
Foreign Gene ◽  
Akabane Virus ◽  
Sense Strand ◽  
Green Fluorescent

ABSTRACTWe generated a recombinant Akabane virus (AKAV) expressing enhanced green fluorescence protein (eGFP-AKAV) by using reverse genetics. We artificially constructed an ambisense AKAV S genome encoding N/NSs on the negative-sense strand, and eGFP on the positive-sense strand with an intergenic region (IGR) derived from the Rift Valley fever virus (RVFV) S genome. The recombinant virus exhibited eGFP fluorescence and had a cytopathic effect in cell cultures, even after several passages. These results indicate that the gene encoding eGFP in the ambisense RNA could be stably maintained. Transcription of N/NSs and eGFP mRNAs of eGFP-AKAV was terminated within the IGR. The mechanism responsible for this appears to be different from that in RVFV, where the termination sites for N and NSs are determined by a defined signal sequence. We inoculated suckling mice intraperitoneally with eGFP-AKAV, which resulted in neurological signs and lethality equivalent to those seen for the parent AKAV. Fluorescence from eGFP in frozen brain slices from the eGFP-AKAV-infected mice was localized to the cerebellum, pons, and medulla oblongata. Our approach to producing a fluorescent virus, using an ambisense genome, helped obtain eGFP-AKAV, a fluorescent bunyavirus whose viral genes are intact and which can be easily visualized.IMPORTANCEAKAV is the etiological agent of arthrogryposis-hydranencephaly syndrome in ruminants, which causes considerable economic loss to the livestock industry. We successfully generated a recombinant enhanced green fluorescent protein-tagged AKAV containing an artificial ambisense S genome. This virus could become a useful tool for analyzing AKAV pathogenesis in host animals. In addition, our approach of using an ambisense genome to generate an orthobunyavirus stably expressing a foreign gene could contribute to establishing alternative vaccine strategies, such as bivalent vaccine virus constructs, for veterinary use against infectious diseases.

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 Poor Glycemic Control Can Increase the Plasma Kidney Injury Molecule-1 Concentration in Normoalbuminuric Children and Adolescents with Diabetes Mellitus

Children ◽  
2021 ◽  
Vol 8 (5) ◽  
pp. 417
Author(s):  
Moon Bae Ahn ◽  
Kyoung Soon Cho ◽  
Seul Ki Kim ◽  
Shin Hee Kim ◽  
Won Kyoung Cho ◽  
...  
Keyword(s):  
Glycosylated Hemoglobin ◽  
Significant Risk ◽  
Area Under The Curve ◽  
Kidney Injury ◽  
Significant Risk Factor ◽  
Controlled Study ◽  
Neutrophil Gelatinase Associated Lipocalin ◽  
Diabetic Children ◽  
Kidney Injury Molecule 1 ◽  
Neutrophil Gelatinase

Diabetic nephropathy (DN) is a serious microvascular complication in childhood diabetes and microalbuminuria has been a solid indicator in the assessment of DN. Nevertheless, renal injury may still occur in the presence of normoalbuminuria (NA) and various tubular injury biomarkers have been proposed to assess such damage. This case-controlled study aimed to evaluate plasma and urinary neutrophil gelatinase-associated lipocalin and kidney injury molecule-1 (KIM-1) levels in diabetic children particularly in those with normo- and high-NA stages and determine their role in predicting DN. Fifty-four children/adolescents with type 1 and 2 diabetes and forty-four controls aged 7–18 years were included. The baseline clinical and laboratory characteristics including plasma and urinary biomarkers were compared. The plasma KIM-1 levels were significantly higher in diabetic children than in the controls and in high-NA children than normo-NA children. Glycosylated hemoglobin (HbA1c) was identified as a significant risk factor for increased plasma KIM-1. The optimal cutoff for HbA1c when the plasma KIM-1 was > 23.10 pg/mL was 6.75% with an area under the curve of 0.77. For diabetic children with mildly increased albuminuria, the plasma KIM-1 complementary to MA may help increase the yield of detecting DN. Our findings also suggested an HbA1c cutoff of 6.75% correlated with increased plasma KIM-1.

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