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Transfection, Spinfection, Exofection, and Luciferase Assays for Analysis of CCN Genes Expression Mechanism

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CCN Proteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2582))

Abstract

Cell communication network factor 2 (CCN2), also known as connective tissue growth factor (CTGF), is protein inducible in response to TGFβ/Smad signal or the transcriptional activity of matrix metalloproteinase 3 (MMP3). We discovered that MMP3 in exosomes is transferable to recipient cells and then translocates into cell nuclei to transactivate the CCN2/CTGF gene. Exosomes and liposomes enable molecular transfection to recipient cells in vitro and in vivo. These small vesicles are surrounded by lipid membranes and carry proteins, RNA, DNA, and small chemicals. Here we define the exosome-based transfection as “exofection.” In addition, spinfection increases the efficiencies of transfection, exofection, and viral infection, thus being compatible with various molecular transfer protocols. Here, we provide protocols, tips, and practical examples of transfection, spinfection, exofection, fluorescence microscopy, and luciferase assays to analyze the CCNs gene expression mechanisms.

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Acknowledgments

T.E. was supported by JSPS Kakenhi, grant numbers 17K11642-TE, 20K09904-CS, 19H03817-MT, 20H03888-HN, 20K20611-MT, 20H03888-HN, 21H03119-TY, and 21K08902-HY. Y.O. was supported by JSPS Overseas Research Fellowship. E.A.T. was supported by the Egypt-Japan Education Partnership Grant. Y.L was supported by Rotary Yoneyama Memorial Foundation Fellowship. Figure 1 was generated using BioRender. The author thanks Shiro Fukuoka, Kazuko Satoshi Kubota, Kuniaki Okamoto, Kisho Ono, Mona Sheta, Chiharu Sogawa, Masaharu Takigawa, and Manh Tien Tran for useful information, materials, discussion, or experimentation.

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Authors and Affiliations

  1. Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan

    Takanori Eguchi & Yanyin Lu

  2. Department of Dental Anesthesiology and Special Care Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan

    Yanyin Lu

  3. Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan

    Eman A. Taha

  4. Department of Biochemistry, Ain Shams University Faculty of Science, Cairo, Egypt

    Eman A. Taha

  5. Division of Molecular and Cellular Biology, Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA

    Yuka Okusha

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  1. Takanori Eguchi
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  2. Yanyin Lu
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  3. Eman A. Taha
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  4. Yuka Okusha
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Corresponding author

Correspondence to Takanori Eguchi .

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Editors and Affiliations

  1. Advanced Research Center for Oral and Craniofacial Sciences, Okayama University Dental School/Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan

    Masaharu Takigawa

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Eguchi, T., Lu, Y., Taha, E.A., Okusha, Y. (2023). Transfection, Spinfection, Exofection, and Luciferase Assays for Analysis of CCN Genes Expression Mechanism. In: Takigawa, M. (eds) CCN Proteins. Methods in Molecular Biology, vol 2582. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2744-0_9

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  • DOI: https://doi.org/10.1007/978-1-0716-2744-0_9

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2743-3

  • Online ISBN: 978-1-0716-2744-0

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