A Comparison of Ypet and Firefly Luciferase as Reporter Proteins for High-Throughput Screening

Yoon, Sei Mee; Namkung, W.; Lee, Jinu

doi:10.1271/bbb.130537

Cited by 4 publications

(3 citation statements)

References 16 publications

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“…This finding is consistent with a number of studies that demonstrated slower kinetics and sensitivity for fluorescent proteins (i.e. Ypet, GFP, DsRed) when compared to Firefly luciferases 9,29,41 . Unlike luciferases, fluorescent proteins require maturation after folding, resulting in slower kinetics 42,43 .…”

Section: Discussionsupporting

confidence: 91%

Reporter gene comparison demonstrates interference of complex body fluids with secreted luciferase activity

Neefjes

Housmans

Akker

et al. 2021

Sci Rep

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Reporter gene assays are widely used to study cellular signaling and transcriptional activity. Few studies describe the use of reporter genes for studying cellular responses on complex body fluids, such as urine and blood. Selection of the optimal reporter gene is crucial for study outcome. Here, we compared the characteristics of five reporter genes (Firefly luciferase, stable- and unstable Nano luciferase, secretable Gaussia luciferase and Red Fluorescent Protein) to study complex body fluids. For this comparison, the NFκB Response Element (NFκB-RE) and Smad Binding Element (SBE) were identically cloned into the five different reporter vectors. Reporter characteristics were evaluated by kinetic and concentration–response measurements in SW1353 and HeLa cell lines. Finally, reporter compatibility with complex body fluids (fetal calf serum, knee joint synovial fluid and human serum) and inter-donor variation were evaluated. Red Fluorescent Protein demonstrated poor inducibility as a reporter gene and slow kinetics compared to luciferases. Intracellularly measured luciferases, such as Firefly luciferase and Nano luciferase, revealed good compatibility with complex body fluids. Secreted Gaussia luciferase appeared to be incompatible with complex body fluids, due to variability in inter-donor signal interference. Unstable Nano luciferase demonstrated clear inducibility, high sensitivity and compatibility with complex body fluids and therefore can be recommended for cellular signaling studies using complex body fluids.

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Section: Discussionsupporting

confidence: 91%

Reporter gene comparison demonstrates interference of complex body fluids with secreted luciferase activity

Neefjes

Housmans

Akker

et al. 2021

Sci Rep

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“…For cryo-histology experiments we generated FVCYpet and FVC GFP that express fluorescent proteins in the cytoplasm of infected cells. For producing FVCYpet, we first generated a full-length replication competent MLV in pLRB303 backbone expressing the fluorescent protein Ypet (FrMLVYpet) inserted after the envelope ORF under a modified IRES (6ATRI) using a strategy described earlier ( Alberti et al., 2015 , Logg et al., 2001 , Nguyen and Daugherty, 2005 , Yoon et al., 2013 ). FVCYpet was made by co-transfecting HEK293 cells with equal amounts of plasmid DNA encoding FrMLVYpet and HindIII fragment released from pBR322 plasmid encoding SFFV LS strain (gift from Frank Malik and Leonard Evans).…”

Section: Methodsmentioning

confidence: 99%

A Protective Role for the Lectin CD169/Siglec-1 against a Pathogenic Murine Retrovirus

Uchil

Haugh

et al. 2019

Cell Host & Microbe

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SummaryLymph- and blood-borne retroviruses exploit CD169/Siglec-1-mediated capture by subcapsular sinus and marginal zone metallophilic macrophages for trans-infection of permissive lymphocytes. However, the impact of CD169-mediated virus capture on retrovirus dissemination and pathogenesis in vivo is unknown. In a murine model of the splenomegaly-inducing retrovirus Friend virus complex (FVC) infection, we find that while CD169 promoted draining lymph node infection, it limited systemic spread to the spleen. At the spleen, CD169-expressing macrophages captured incoming blood-borne retroviruses and limited their spread to the erythroblasts in the red pulp where FVC manifests its pathogenesis. CD169-mediated retroviral capture activated conventional dendritic cells 1 (cDC1s) and promoted cytotoxic CD8+ T cell responses, resulting in efficient clearing of FVC-infected cells. Accordingly, CD169 blockade led to higher viral loads and accelerated death in susceptible mouse strains. Thus, CD169 plays a protective role during FVC pathogenesis by reducing viral dissemination to erythroblasts and eliciting an effective cytotoxic T lymphocyte response via cDC1s.

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“…Firefly luciferases have also been expressed in live mammals to visualize disease states [20–23], tumor growth and metastasis [24–27], cell trafficking [28, 29] and genetic regulation [30]. Many of these applications can be applied in high-throughput screening format in support of drug development [31–34]. One of the distinctive properties of the beetle luciferases is the highly specific dependence of the Luc 1 -catalyzed light emission process on ATP (Eqs (1–3)).…”

Section: Introductionmentioning

confidence: 99%

An enhanced chimeric firefly luciferase-inspired enzyme for ATP detection and bioluminescence reporter and imaging applications

Branchini

Southworth

Fontaine

et al. 2015

Analytical Biochemistry

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Firefly luciferases, which emit visible light in a highly specific ATP-dependent process, have been adapted for a variety of applications including gene reporter assays, whole-cell biosensor measurements and in vivo imaging. We have previously reported the ~2-fold enhanced activity and 1.4-greater bioluminescence quantum yield properties of a chimeric enzyme that contains the N-domain of Photinus pyralis luciferase joined to the C-domain of Luciola italica luciferase. Subsequently, we identified 5 amino acid changes based on L. italica that are the main determinants of the improved bioluminescence properties. Further engineering to enhance thermal and pH stability produced a novel luciferase called PLG2. We present here a systematic comparison of the spectral and physical properties of the new protein with P. pyralis luciferase and demonstrate the potential of PLG2 for use in assays based on the detection of femtomol levels of ATP. Additionally, we compared the performance of a mammalian codon-optimized version of the cDNA for PLG2 with the luc2 gene in HEK293T cells. Using an optimized low-cost assay system, PLG2 activity can be monitored in mammalian cell lysates and in living cells offering an improved alternative to Promega’s luc2 for reporter and imaging applications.

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A Comparison of Ypet and Firefly Luciferase as Reporter Proteins for High-Throughput Screening

Cited by 4 publications

References 16 publications

Reporter gene comparison demonstrates interference of complex body fluids with secreted luciferase activity

Reporter gene comparison demonstrates interference of complex body fluids with secreted luciferase activity

A Protective Role for the Lectin CD169/Siglec-1 against a Pathogenic Murine Retrovirus

An enhanced chimeric firefly luciferase-inspired enzyme for ATP detection and bioluminescence reporter and imaging applications

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