Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021:2274:79-87.
doi: 10.1007/978-1-0716-1258-3_8.

A Split-Luciferase-Based Cell Fusion Assay for Evaluating the Myogenesis-Promoting Effects of Bioactive Molecules

Qiaojing Li  1 , Hideaki Yoshimura  2 , Takeaki Ozawa  1
Affiliations

A Split-Luciferase-Based Cell Fusion Assay for Evaluating the Myogenesis-Promoting Effects of Bioactive Molecules

Qiaojing Li et al. Methods Mol Biol. 2021.

Abstract

A split-luciferase-based cell fusion assay enables high-throughput screening of myogenesis-promoting chemicals in chemical libraries. The assay consists of two C2C12 myoblast-derived cell lines (N- and C-cells), each of which stably expresses either an N- or C-terminal split-firefly luciferase (FLuc) fragment fused to a naturally split DnaE intein (N- and C-probes, respectively). The fusion of N- and C-cells during myogenesis induces bioluminescence (BL) in the cytosol due to a stable reconstitution of the split-FLuc. Thus, the myogenesis-promoting effects of a chemical compound can be determined through the enhanced BL intensity. Here, we describe the preparation of N- and C-cells and determination of the myogenesis-promoting effects of imatinib using a 96-well microplate-based assay.

Keywords: 96-Well microplate-based assay; C2C12 myoblast; Cell fusion; DnaE intein; Firefly luciferase; Imatinib; Myogenesis; Protein splicing; Split luciferase reconstitution.

PubMed Disclaimer

References

    1. Ozawa T, Kaihara A, Sato M, Tachihara K, Umezawa Y (2001) Split luciferase as an optical probe for detecting protein-protein interactions in mammalian cells based on protein splicing. Anal Chem 73:2516–2521 - DOI
    1. Yoshimura H, Ozawa T (2014) Methods of split reporter reconstitution for the analysis of biomolecules. Chem Rec 14:492–501 - DOI
    1. Kanno A, Yamanaka Y, Hirano H, Umezawa Y, Ozawa T (2007) Cyclic luciferase for real-time sensing of caspase-3 activities in living mammals. Angew Chem Int Ed Engl 46:7595–7599 - DOI
    1. Wu H, Hu Z, Liu XQ (1998) Protein trans-splicing by a split intein encoded in a split DnaE gene of Synechocystis sp. PCC 6803. Biochemistry 95:9226–9231
    1. Li Q, Yoshimura H, Komiya M, Tajiri K, Uesugi M, Hata Y, Ozawa T (2018) A robust split-luciferase-based cell fusion screening for discovering myogenesis-promoting molecules. Analyst 143:3472–3480 - DOI

LinkOut - more resources