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J. Virol. 88, 2034–2046. Stable, high-level expression of reporter proteins from improved alphavirus expression vectors to track replication and dissemination during encephalitic and arthritogenic disease. 2014

Sun, C., Gardner, C.L., Watson, A.M., Ryman, K.D. and Klimstra, W.B.

Notes: Researchers were interested in using a reporter gene that could be stably expressed in alphaviruses without attenuating infectivity. cDNA clones of eastern equine encephalitis (EEEV), Venezuelan equine encephalitis (VEEV), Sindbis (SINV) and chikungunya (CHIKV) viruses had either firefly luciferase or a FLAG-tagged NanoLuc™ luciferase genes inserted in the genomes using three different insertion points. The cDNAs were then transcribed to generate infectious viral RNAs that were then electroporated into BHK-21 cells, virus particles harvested from the supernatant after 18–24 hours and stored at –80°C in single-use aliquots as viral stock.

To assess how the reporter genes affected viral replication, BHK-21 cells were infected at a multiplicity of infection (MOI) of 0.1 PFU/cell or 5 PFU/cell. After 1 hour, cells were washed and medium replaced. At time points 0, 6, 12, 18, 24 and 48 hours, supernatant was sampled for plaque assay titration and cells lysed with 1X Passive Lysis Buffer for measuring reporter activity using the Luciferase Assay System for firefly luciferase or Nano-Glo® Luciferase Assay for NanoLuc™ luciferase.

To examine how the presence of the reporter gene might affect viral infectivity over time, BHK-21 cells were infected with SINV reporter viruses at an MOI of 0.1 PFU/cell and passage 1 (P1) viral stock was harvested 18 hours after infection. The SINV virus was then diluted 1:1,000 for infection of fresh cells, serially passaged nine more times. Supernatants from P1– P10 viruses were titrated by plaque assay; cells were lysed with 1X Passive Lysis Buffer to assay luciferase activity. Parallel protein lysates were prepared with whole-cell extract lysis buffer for Western blotting analysis using Anti-Luciferase pAb for firefly luciferase and an anti-FLAG antibody for NanoLuc™ luciferase.

Five-day-old CD-1 mice were infected with 1,000 PFU of SINV reporter viral stock in the ventral thorax region while six to eight-week-old CD-1 mice were infected with 1,000 PFU of EEEV reporter viral stock in the right rear footpad, and monitored for at least ten days. Groups of mice were sacrificed at various intervals, tissues (e.g., brain and spleen) homogenized in 1X Passive Lysis Buffer, frozen at –80°C and luciferase activity analyzed. For imaging studies, adult C57Bl/6 IFNAR1-/- mice were infected in both hind limb footpads with 1,000 PFU of either firefly or NanoLuc™ luciferase-TaV viral stocks. Six, 24 or 48 hours post-infection, 3mg of D-luciferin or 10μg of furimazine were injected into the tail vein. Mice were imaged for 2 seconds within 2 minutes of substrate administration. (4442)

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Am. J. Physiol. Cell Physiol. 287, 1031-1040. Molecular analysis of fiber type-specific expression of murine myostatin promoter. 2004

Salerno, M.S., Thomas, M., Forbes, D., Watson, T., Kambadur, R., and Sharma, M.

Notes: In this article, a 2.5- and a 1.7-kb fragment of the myostatin promoter were amplified by PCR and cloned into the pGEM®-T Easy vector.  These clones were then subcloned into the pGL3-Basic Vector.  Deletion mutants of the constructs were made and used to transient transfect mouse muscle C2C12 cells.  The Luciferase Assay System was then used to analyze transfectants.  The researchers also injected luciferase-reporter constructs into mouse muscle.  Luciferase activity in the mouse muscle was examined by grinding isolated muscles in liquid nitrogen and resuspending them in Cell Culture Lysis Buffer.  Ten micron cryosections of the muscles were also used in immunohistochmical staining experiments.  For these experiments, the researchers used a 1:50 dilution of Promega’s polyclonal anti-luciferase antibody, a secondary antibody and tertiary fluorescein-labeled conjugate.  The slides were counter stained with DAPI. (3147)

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J. Biol. Chem. 277, 19511-19520. A common mechanism of stage-regulated gene expression in Leishmania mediated by a conserved 3´-untranslated region. 2002

Boucher, N., Wu, Y., Dumas, C., Dube, M., Sereno, D., Breton, M. and Papadopoulou, B.

Notes: In this paper Promega's Anti-Luciferase pAb was used in western blot analysis of Leishmania cell extracts. The antibody was used at a 1:2,000 dilution, and was incubated for 90 minutes with the membrane. (2639)

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J. Biol. Chem. 277, 46374-46384. Analysis of transcriptional regulatory sequences of the N-methyl-D-aspartate receptor 2A subunit gene in cultured cortical neurons and transgenic mice. 2002

Desai, A., Turetsky, D., Vasudevan, K. and Buonanno, A.

Notes: In this paper Promega's Anti-Luciferase pAb was used in immunocytochemistry to detect luciferase expression in cultured cortical neurons transfected with various constructs. The antibody was used at a 1:500 dilution in TSA blocking buffer and was incubated with the cells overnight at 4°C. (2640)

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Am. J. Physiol. Cell Physiol. 279, C257-65. Tenotomy decreases reporter protein synthesis via the 3'-untranslated region of the beta-myosin heavy chain mRNA. 2000

Ashley, W.W. Jr, and Russell, B.

Notes: In a rat model of muscle atrophy, the myosin heavy chain 3'-untranslated region mediates a decrease in myosin heavy chain expression. To quantitate this effect, luciferase was placed under the control of either the myosin heavy chain or SV40 3'-UTR in the pGL3 Control Vector to show that luciferase expression decreased 63% in this model compared to controls. Luciferase assay were performed using one of Promega's luciferase substrates (not specified). For luciferase assays, soleus muscle was ground to a fine powder in liquid nitrogen in a cell lysis buffer (0.5% Nonidet® P-40 (NP-40), 10 mM HEPES, 3 mM MgCl2, 40 mM KCl, 2 mM dithiothreitol (DTT), 0.5 mM PMSF, and a protease inhibitor cocktail. The tissue was homogenized on ice, centrifuged at 3,000 rpm at 4°C to remove debris, and stored at 80°C. For immunohistochemistry, tissue was frozen, serial sectioned into 10µm slices, fixed with 4% paraformaldehyde, and stained with the Anti-Luciferase pAb. (2449)

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