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Dual light reporter system

Manufactured by Thermo Fisher Scientific

The Dual Light Reporter system is a lab equipment product that measures the activity of two different reporter genes simultaneously. It provides a quantitative measurement of gene expression by detecting and analyzing the luminescence signals from the reporter genes.

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7 protocols using dual light reporter system

1

Splicing Reporter Assay in SW620 Cells

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The SW620 cells were electroporated with the double-reporter minigene pTN24 splicing reporter plasmid51 (link), treated with doxycycline or DMSO 72 h and harvested. The Dual Light Reporter System (Applied Biosystems) was used to detect reporter expression, followed by calculation of the the luciferase to β-galactosidase ratio.
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2

Splicing Reporter Assay in 293T Cells

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The 293T cells were seeded in a 12-well plate (8 × 104 cells per well). After culturing overnight, cells were transfected with the pTN24 splicing reporter plasmid (containing a constitutively expressed β-galactosidase reporter for transfection normalization and a luciferase reporter that is conditional on removal of a translational stop codon by splicing). In some experiments, cells were also cotransfected with PTEN-specific siRNA or siRNA scramble control. Cells were harvested 48 h after transfection, and reporter expression was detected as previously described62 (link) using the Dual Light Reporter System (Applied Biosystems) and analyzed by calculating the ratio of the luciferase to β-galactosidase signals. Three independent experiments were performed, and the data were analyzed using Student’s t-test.
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3

Axolotl Transcription Factors in NPCs

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B35, neural progenitor cells were co-transfected with pGFAP: Luciferase, β-Galatosidase internal control, axolotl c-Jun and/or JunB subcloned into pCMV:GFP plasmid (Clontech) with Lipofectamine 3000. After 48 h luciferase activity was determined using Dual Light Reporter system (Thermo) according to the manufacturer’s protocol.
The similar experiment was carried out using the axolotl GFAP promoter.
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4

Wnt Signaling in HEK293T Cells

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Fzd1/2/4/5/7/8-knockout HEK293T cells (69 (link)) were maintained in DMEM supplemented with 10% v/v fetal bovine serum. Cells were seeded into white 96-well plates (PerkinElmer) and transfected with 80 ng SuperTopFlash plasmid (laboratory of Dr. Randall Moon; Addgene plasmid #12456), 20 ng CMV-driven LacZ plasmid, and 1 ng FLAG-tagged Fzd4 or FZD5 plasmid per well using Lipofectamine 2000 according to the manufacturer’s instructions (ThermoFisher). Purified xWnt8 or Norrin (with MBP tag removed as described above for BLI) were added 24 h post-transfection and cells were incubated for 16–18 h with ligand. Cells were lysed and luciferase and β-galactosidase signals were quantified on a BioTek Synergy 2 plate reader using the Dual-Light Reporter System (ThermoFisher) according to the manufacturer’s instructions.
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5

Transcriptional Regulation by DUX4 and Pax7

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HEK-293 or NIH-3T3 cells (12,500 cells/well) were transfected with pMSCV-IRES-eGFP plasmids encoding DUX4 and/or Pax7, or dominant negative DUX4-ERD, or dominant negative Pax7-ERD15 (link), 29 (link), together with reporter plasmids encoding either DUX4-responsive promoters driving luciferase expression (pZSCAN4-luc35 (link) or RFPL4B-luc) or a Pax3/7-responsive element driving β-galactosidase (p34 plasmid15 (link)) using Lipofectamin LTX (Thermofisher). A pRSV vector encoding either β-galactosidase or luciferase as appropriate was co-transfected as an internal control for normalisation of transfection efficiency. Total amount of DNA transfected per reaction was 1.5 mg, in equal ratios for reporters and plasmids encoding genes of interest, and 250 ng/reaction of the internal normaliser vector. Cells were harvested 24 h after transfection, and assayed using the Dual-light Reporter system (Thermofisher) according to the manufacturer’s instructions. 3-4 independent transfections were performed in technical triplicate. Reporter gene activity was measured using Glomax-Multi + plate reader (Promega) and normalised for transfection efficiency. N = 3/4 for each cell line, analysis of variance (ANOVA) revealed significant intensity differences, post hoc unpaired two-tailed t-tests were employed to assess significant pairwise differences.
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6

Axolotl c-Jun 3' UTR Luciferase Assay

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3’ UTR luciferase experiments
B35 neural progenitor cells were plated in a 96 well plate (Celltreat Scientific Products) at a concentration of 1.0 × 105 cells/mL and allowed to adhere overnight. The next day cells were co-transfected with axolotl c-Jun 3’ UTR luciferase reporter, β-Galatosidase internal control and 100 nM of miR-200a or control mimic (Qiagen) Lipofectamine 3000 (Invitrogen). After 48 h luciferase activity was determined using Dual Light Reporter system (Thermo) according to the manufacturers protocol.
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7

DUX4-driven Transcriptional Regulation

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C2C12 myoblasts were co-transfected using Lipofectamine LTX (Thermofisher) with DUX4c or DUX4 constructs or control GFP and DUX4-responsive promoters driving luciferase (pZSCAN4-luc, pKHDC1L-luc, RFPL4b-luc), together with a pRSV-β-galactosidase construct to normalise transfections. Myoblasts were harvested 24 h later, and assayed using the Dual-light Reporter system (Thermofisher) in three transfections measured in triplicate on a Glomax-Multi+ plate reader (Promega).
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