Notes: Antisense fluorophore octaarginine peptide nucleic acid (PNA) conjugates are investigated cellular activity using a photochemical internalization (PCI) drug delivery method. PCI utilizes light to generate reactive oxygen species that can damage the endosomal membrane and lead to drug release. Specifically, cellular localization and antisense activity was monitored for two conjugates, tetramethylrhodamine and Alexa Fluor 555. Luminescence and cell viability were measured using the Bright-Glo™ Assay and CellTiter-Glo® Assay, respectively. (5172)

Notes: The presence of post-transcriptional modifications (PTMs) in viral RNAs is investigated using mass spectrometry. A wide-range of PTMs on RNAs from Zika virus, Dengue virus, HIV-1, Poliovirus, and hepatitis C virus were identified. Additionally, PTMs on cellular RNAs were measured in response to viral infection and stress response. The Dual-Luciferase® Reporter Assay was used to monitor cellular stress. (5091)

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)

Notes: These authors studied KLF6 expression during human trophoblast cell differentiation, and its role in the regulation of genes associated with placental development and pregnancy maintenance. They used immunofluorescence microscopy, RT-qPCR and luciferase reporter assays to investigate cellular localization, mRNA expression, and transcriptional activation. Reporter assays were performed using various luciferase reporter constructs, the Dual-Luciferase^® Assay, and the GloMax^®-Multi Detection System. KLF6 was shown to play a role as transcriptional regulator of relevant genes for placental differentiation and physiology such as βhCG and PSG. (4197)

Notes: Telomeres shorten by 50–100 bases with each cell division, making the telomere a "mitotic counter" that can limit cellular lifespan. Telomerase is a two-component protein consisting of a reverse transcriptase (hTERT) bound to its own RNA template that can act to maintain telomere length in dividing cells. Telomerase is highly active in dividing cells such as germ cells, stem cells and many cancers. This paper investigated the role of methylation of the hTERT promoter and the transcription factor CTCF in regulation of telomerase activity. LacZ reporter plasmids driven by the hTERT minimal promoter were transiently transfected into HeLa cells, and reporter assays were performed on lysate generated using Passive Lysis Buffer. The hTERT minimal promoter did not show activity if all of the CpG sites were methylated. The promoter and first exon of hTERT were amplified using PCR Master Mix from sodium bisulfite-treated genomic DNA isolated from telomerase-positive cell lines and tissues. The resulting fragments were cloned using the pGEM^®-T Vector System II. For the methylation cassette assay, methylated and unmethylated fragments were cloned into a methylated or unmethylated vector using the LigaFast™ Rapid DNA Ligation System. The authors conclude that methylation plays a dual role in regulating hTERT expression. CTCF will bind to the first exon of hTERT when the hTERT CpG island is not methylated, resulting in downregulation of hTERT expression. Although CTCF cannot bind the hTERT promoter when the DNA is completely methylated, the methylation itself completely represses transcription. In situations where there is partial methylation of the promoter, such as in tumor cells, CTCF cannot bind to the promoter, but the partial methylation is not enough to repress transcription, and hTERT is expressed. (3641)

Notes: A key component in the pathogenesis of Alzheimer's disease is cerebral accumulation of amyloid-beta protein (Aβ). Aβ is produced by proteolysis of amyloid-β-protein precursor (APP) by ß- and gamma-secretases, thus these enzymes are considered important drug targets for Alzheimer's disease. Existing assays for assessing inhibition of alpha-, beta- and gamma-secretases include HPLC or ELISA assays that are cumbersome, expensive and not well-suited to high-throughput screening. The authors developed a luciferase reporter system to identify new molecules that inhibit APP processing. They then successfully interfaced this sensitive, specific and quantitative assay with a high-throughput screen, useful for identifying both inhibitors and stimulators of APP processing. (3775)

Notes: The firefly and Renilla luciferase coding regions were amplified from the pGL3 and pRL-CMV Vectors and cloned into various yeast expression vectors. Strains of Saccharomyces cerevisiae were transformed with these constructs and analyzed with the Dual-Luciferase^® Reporter Assay System. The authors created yeast lysates for the Dual-Luciferase^® Reporter Assay System using 1X Passive Lysis Buffer. Several factors important to assay performance as well as firefly and Renilla luciferase expression were explored, including the stability of both luciferases stored in lysates at room temperature for various periods of time, optimal culture density before lysis of transformants and the firefly luciferase half-life in S. cerevisiae. (3298)

Notes: Researchers cloned the Photinus and Renilla luciferase ORFs into the pSP64 Poly(A) Vector to create a dual-reporter vector named SP6P. A similar vector, SP6R.4G(-508/-3).P, was created in which a 5´ untranslated region from the Saccharomyces cerevisiae TIF4631 gene was cloned between the two reporter genes. These two vectors were used to transform yeast strains. The resultant transformants were lysed using Passive Lysis Buffer and a modified lysis procedure.   Lysates were analyzed for luciferase activities using the Dual-Luciferase^® Reporter Assay System and a TD20/20 luminometer. The researchers also cloned and sequenced the 5´  untranslated region of TIF4631 by using a RACE-PCR technique followed by cloning the PCR amplimers into the pGEM^®-T Vector. (2845)

Notes: To study response to the thyroid hormone (T₃), a firefly luciferase construct was made with a T₃ responsive element upstream of a thymidine kinase minimal promoter. This plasmid and the Renilla luciferase reporter plasmid phRL-SV40 were injected into the dorsal muscle of NF55 stage Xenopus tadpoles. One microliter of solution containing between 0.7 and 2.1μg of DNA in 0.07M NaCl with a tracking dye were injected. After two days, the dorsal muscles were collected, flash frozen and sonicated in Passive Lysis Buffer. This homogenate was assayed for luciferase activity using the Dual-Luciferase® Reporter Assay System. (2762)

Notes: The ability of a 5' UTR to act as an internal ribosome entry site (IRES) was explored in this study. The 5' UTR of YAP1 and p150 was cloned between the Renilla and firefly luciferase genes driven by a GAL1 promoter. The IRES effect was examined by measuring firefly luciferase expression driven by the IRES normalized against the first cistron, Renilla luciferase. To use the Dual-Luciferase^® Assay in yeast, the liquid culture was pelleted then resuspended in Passive Lysis Buffer. Glass beads were added to the mixture, which was then and vortexed using two, 30-second pulses. This mix was then harvested at high speed and 20μl of the supernatant was used in the Dual-Luciferase^®Assay. (3032)

Notes: The pTARGET™ Mammalian Expression Vector was used to clone and express a portion of the ARA9, a protein that interacts with the aryl hydrocarbon receptor. The expressed protein was used in a two-hybrid assay using a GAL4-fusion protein containing a portion of the aryl hydrocarbon receptor. Interaction of the two proteins is reported by activation of the luciferase in the pG5-luc Vector, which is a portion of the CheckMate™ Mammalian Two-Hybrid System. The two-hybrid studies were performed in COS-1 cells and were controlled with a cotransfection with a β-galactosidase vector. Cells were lysed with the Passive Lysis Buffer and luciferase activities determined with the Luciferase Assay System. (0842)