Pmcs gaussia luc vector

Manufactured by Thermo Fisher Scientific

Sourced in United States

The PMCS-Gaussia Luc Vector is a laboratory tool used for gene expression analysis. It contains the Gaussia luciferase reporter gene, which can be used to measure gene expression levels in various cell lines and experimental models. The vector provides a convenient way to quantify gene activity through luminescence measurements.

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Lab products found in correlation

Gaussia luciferase glow assay kit, by Thermo Fisher Scientific (1 mentions) Attractene, by Qiagen (1 mentions) Abi prism 3100 genetic analyzer, by Thermo Fisher Scientific (1 mentions) Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (1 mentions) Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions) Q5 site directed mutagenesis kit, by New England Biolabs (1 mentions) Glomax luminometer, by Promega (1 mentions) Agilent rna 6000 nano assay, by Agilent Technologies (1 mentions) Rneasy mini kit, by Qiagen (1 mentions) 2100 bioanalyzer, by Agilent Technologies (1 mentions)

4 protocols using pmcs gaussia luc vector

Regulatory Region of Mouse Tspo Gene

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The regulatory region of the mouse Tspo gene was amplified using forward primers and a common reverse primer with the addition of the Spe I (forward) or Bam HI (reverse) sites as described in S1 Table. The products were ligated to the pMCS-Gaussia Luc Vector (Thermo Fisher Scientific), and the sequences were confirmed via sequencing (S1 Table) using a BigDye® Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific); the reaction products were then purified using a Gel Filtration Cartridge (Edge Bio, San Jose, CA, USA). The sample sequences were analyzed using an ABI PRISM® 3100 Genetic Analyzer (Applied Biosystems, Waltham, MA, USA).
Various constructs were transfected into BV-2 cells using Attractene (QIAGEN, Hilden, Germany) in accordance with the manufacturer’s instructions. A reporter gene assay was performed using a Gaussia Luciferase Glow Assay Kit (Thermo Fischer Scientific) according to the manufacturer’s instructions.

Shimoyama S., Furukawa T., Ogata Y., Nikaido Y., Koga K., Sakamoto Y., Ueno S, & Nakamura K. (2019). Lipopolysaccharide induces mouse translocator protein (18 kDa) expression via the AP-1 complex in the microglial cell line, BV-2. PLoS ONE, 14(9), e0222861.

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Secreted Luciferase Promoter Constructs

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Secreted promoter luciferase constructs were generated as depicted in Fig. 1D. The wild-type FUK promoter was subcloned from WM793 cDNA into the pMCS–Gaussia Luc vector (Thermo Fisher Scientific) using Eco RI and Hind III restriction enzyme sites. ATF2 binding site mutant promoter constructs (E1, E2, and E3) were generated using the Lightning Mutagenesis Kit (Agilent) according to the manufacturer’s protocols. Primers are provided in table S1.
Cells were transfected as indicated, and media samples were collected and measured for Gaussia and Cypridina luciferase activity using a standard luminometer. Gaussia luciferase activity values were normalized to Cypridina luciferase activity values (from pCMV–Cypridina luciferase construct, which exhibits a constitutively cytomegalovirus (CMV) promoter–driven secreted Cypridina luciferase). The Cypridina luciferase–normalized values were plotted relative to the values indicated in the figure legends. The data are representative of three independent experiments.

Lau E., Feng Y., Claps G., Fukuda M.N., Perlina A., Donn D., Jilaveanu L., Kluger H., Freeze H.H, & Ronai Z.A. (2015). The transcription factor ATF2 promotes melanoma metastasis by suppressing protein fucosylation. Science signaling, 8(406), ra124.

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Androgen Receptor Regulation of FUT4 Promoter

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The 277-bp wild-type FUT4 promoter region (SwitchGear Genomics) containing the putative AR-binding element (5’-AAACATTTTGTTCTC-3’) was cloned from WM793 cDNA into the pMCS-Gaussia Luc vector (ThermoFisher Scientific) between the SacI and BamHI restriction enzyme sites. ARE mutant promoter constructs (S1, S2, and S3) were generated using the Q5 Site-Directed Mutagenesis Kit (NEB) per the manufacturer’s protocols. Sequence verification was performed by Eton Bioscience. Primers were synthesized by IDT and listed in Supplementary Data 1.
Melanoma cells were co-transfected with EV or wild-type/mutant FUT4 promoter-Gaussia luciferase constructs and pCMV-Cypridina (constitutive control) vector (ThermoFisher Scientific) at a ratio of 50:1 using Lipofectamine 3000 (Invitrogen). At 24 h after transfection, cells were treated either with methanol/DHT or with DMSO/ARi. At 48 h after treatment, samples of media were collected and subject to Pierce Gaussia/Cypridina Luciferase Flash Assay (ThermoFisher Scientific) per the manufacturer’s protocols. Luciferase activity was measured using a Promega GloMax Luminometer. Gaussia luciferase values were normalized to Cypridina values to control for the transfection efficiency.

Liu Q., Adhikari E., Lester D.K., Fang B., Johnson J.O., Tian Y., Mockabee-Macias A.T., Izumi V., Guzman K.M., White M.G., Koomen J.M., Wargo J.A., Messina J.L., Qi J, & Lau E.K. (2024). Androgen drives melanoma invasiveness and metastatic spread by inducing tumorigenic fucosylation. Nature Communications, 15, 1148.

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In Vitro Transcription of Therapeutic mRNA

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The in vitro transcription (IVT) vector utilized for producing Gluc mRNA was sub-cloned from pDNAs encoding Gaussia princeps luciferase (pMCS-Gaussia Luc Vector; Cat. 16146, ThermoFisher Scientific Inc., Madison, WI, USA), and the cDNA fragment was inserted into the pSP73 vector (Cat. P2221, Promega Corporation, Madison, WI, USA) under the control of the T7 promoter containing 120 bps and 240 bps chemically synthesized poly(d(A/T) fragments at the downstream of the cDNA region [16 (link),17 (link)] (Figure S1a). Then, the vectors were linearized with BsmBI, blunted with T4 DNA polymerase, purified with gel electrophoresis, and served as templates for IVT using the HiScribe^® T7 High Yield RNA Synthesis Kit (Cat. E2040S, BioLab, Rockville, MD, USA) to generate mRNA, and the cap structure was added using the Vaccinia Capping System (Cat. M2080S, BioLab, USA). The mRNAs encoding BMP2 and TGFβ3 were similarly constructed from the vectors carrying human BMP2 and TGFβ3 ORF sequences (pBac-LrpwpA), which were gifts from Prof. Hu [9 (link)] (Figure S2a). Prior to the experiments, all transcribed mRNAs were purified using the RNeasy Mini kit (Cat. 74004, Qiagen, Venlo, The Netherlands) and analyzed for their size and purity using the Agilent RNA 6000 Nano Assay on a BioAnalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA) (Figures S1b and S2b).

Tsou H.K., Wu C.H., Chan L.Y., Kataoka K., Itokazu N., Tsuzuki M., Hu H., Zhuo G.Y., Itaka K, & Lin C.Y. (2023). Administration of mRNA-Nanomedicine-Augmented Calvarial Defect Healing via Endochondral Ossification. Pharmaceutics, 15(7), 1965.

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