ArticleMethods in molecular biology (Clifton, N.J.)2022
Synthetic Assembly DNA Cloning of Multiplex Hextuple Luciferase Reporter Plasmids.
Article in Methods in molecular biology (Clifton, N.J.), 2022. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 4 papers.
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The trial behind it
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Who cites it
4 citing papers in PubMed.
- A multiplex hextuple luciferase assay for advancing cellular signaling analysis in plant cells.Plant communications · 2026Article
- Going against the family: Perturbation of a greenbeard pathway leads to falsebeard cheating.iScience · 2024Article
- Synthetic Assembly DNA Cloning to Build Plasmids for Multiplexed Transgenic Selection, Counterselection or Any Other Genetic Strategies Using Drosophila melanogaster.Current protocols · 2023Article
- Serial Recombineering Cloning to Build Selectable and Tagged Genomic P[acman] BAC Clones for Selection Transgenesis and Functional Gene Analysis using Drosophila melanogaster.Current protocols · 2023Article
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Authors and funding
3 authors.
Funding
Abstract
Multiplex hextuple luciferase assaying allows monitoring the activity of five experimental pathways against one control at the same time. To perform multiplex hextuple luciferase assaying, six orthogonal luciferase reporter units are needed of which five are pathway-specific and one acts as a control for normalization. To ensure stoichiometric delivery of all six luciferase reporters in every transfected cell, synthetic assembly DNA cloning is used to stitch together all six luciferase reporter units into a single vector. Here, we provide a detailed three-step synthetic assembly DNA protocol to generate multiplex hextuple luciferase reporter plasmids for any five cellular signaling pathways of interest, against a control normalization pathway. A first protocol is provided on how to generate plasmids that contain novel transcription factor-binding motifs for specific transcription factors. A second protocol details on how to couple these novel transcription factor-binding motifs to one of five orthogonal luciferases to obtain specific luciferase reporters for cellular signaling pathways acting upstream of those transcription factor-binding motifs. Finally, a third protocol provides details on how to assemble orthogonal luciferase reporters for five cellular signaling pathways acting upstream of five unique transcription factor-binding motifs together with a control constitutive pathway luciferase reporter that will be used for normalization to obtain a final multiplex hextuple luciferase vector.
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Registered trials
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