A method of producing genetically manipulated mouse mammary gland

Hiroaki Tagaya, Kosuke Ishikawa, Yoshito Hosokawa, Shun Kobayashi, Yukino Ueoka, Mayuna Shimada, Yasuko Ohashi, Hirofumi Mikami, Mizuki Yamamoto, Tatsuya Ihara, Kentaro Kumazawa, Kosuke Sugihara, Naoki Goshima, Shinya Watanabe, Kentaro Senba

    Research output: Contribution to journalArticle

    Abstract

    BACKGROUND: To obtain a deep understanding of the mechanism by which breast cancer develops, the genes involved in tumorigenesis should be analyzed in vivo. Mouse mammary gland can regenerate completely from a mammary stem cell (MaSC), which enables us to analyze the effect of gene expression and repression on tumorigenesis in mammary gland regenerated from genetically manipulated MaSCs. Although lentiviral and retroviral systems have usually been applied for gene transduction into MaSCs, they are associated with difficulty in introducing long, repeated, or transcriptional termination sequences. There is thus a need for an easier and quicker gene delivery system. METHODS: We devised a new system for gene delivery into MaSCs using the piggyBac transposon vectors and electroporation. Compared with viral systems, this system enables easier and quicker transfection of even long, repeated, or transcriptional termination DNA sequences. We designed gene expression vectors of the transposon system, equipped with a luciferase (Luc) expression cassette for monitoring gene transduction into regenerative mammary gland in mice by in-vivo imaging. A doxycycline (Dox)-inducible system was also integrated for expressing the target gene after mammary regeneration to mimic the actual mechanism of tumorigenesis. RESULTS: With this new gene delivery system, genetically manipulated mammary glands were successfully reconstituted even though the vector size was > 200 kb and even in the presence of DNA elements such as promoters and transcription termination sequences, which are major obstacles to viral vector packaging. They differentiated correctly into both basal and luminal cells, and showed normal morphological change and milk production after pregnancy, as well as self-renewal capacity. Using the Tet-On system, gene expression can be controlled by the addition of Dox after mammary reconstitution. In a case study using polyoma-virus middle T antigen (PyMT), oncogene-induced tumorigenesis was achieved. The histological appearance of the tumor was highly similar to that of the mouse mammary tumor virus-PyMT transgenic mouse model. CONCLUSIONS: With this system, gene transduction in the mammary gland can be easily and quickly achieved, and gene expression can be controlled by Dox administration. This system for genetic manipulation could be useful for analyzing genes involved in breast cancer.

    Original languageEnglish
    Number of pages1
    JournalBreast cancer research : BCR
    Volume21
    Issue number1
    DOIs
    Publication statusPublished - 2019 Jan 5

    Fingerprint

    Human Mammary Glands
    Gene Transfer Techniques
    Doxycycline
    Carcinogenesis
    Gene Expression
    Polyomavirus
    Breast
    Viral Tumor Antigens
    Genes
    Breast Neoplasms
    Mouse mammary tumor virus
    Virus Assembly
    Electroporation
    Neoplasm Genes
    Gene Expression Profiling
    Luciferases
    Oncogenes
    Transgenic Mice
    Transfection
    Regeneration

    Keywords

    • Doxycycline
    • Electroporation
    • MaSC
    • PiggyBac
    • Tet-On system
    • Transgenesis
    • Transposon

    ASJC Scopus subject areas

    • Oncology
    • Cancer Research

    Cite this

    A method of producing genetically manipulated mouse mammary gland. / Tagaya, Hiroaki; Ishikawa, Kosuke; Hosokawa, Yoshito; Kobayashi, Shun; Ueoka, Yukino; Shimada, Mayuna; Ohashi, Yasuko; Mikami, Hirofumi; Yamamoto, Mizuki; Ihara, Tatsuya; Kumazawa, Kentaro; Sugihara, Kosuke; Goshima, Naoki; Watanabe, Shinya; Senba, Kentaro.

    In: Breast cancer research : BCR, Vol. 21, No. 1, 05.01.2019.

    Research output: Contribution to journalArticle

    Tagaya, H, Ishikawa, K, Hosokawa, Y, Kobayashi, S, Ueoka, Y, Shimada, M, Ohashi, Y, Mikami, H, Yamamoto, M, Ihara, T, Kumazawa, K, Sugihara, K, Goshima, N, Watanabe, S & Senba, K 2019, 'A method of producing genetically manipulated mouse mammary gland' Breast cancer research : BCR, vol. 21, no. 1. https://doi.org/10.1186/s13058-018-1086-8
    Tagaya H, Ishikawa K, Hosokawa Y, Kobayashi S, Ueoka Y, Shimada M et al. A method of producing genetically manipulated mouse mammary gland. Breast cancer research : BCR. 2019 Jan 5;21(1). https://doi.org/10.1186/s13058-018-1086-8
    Tagaya, Hiroaki ; Ishikawa, Kosuke ; Hosokawa, Yoshito ; Kobayashi, Shun ; Ueoka, Yukino ; Shimada, Mayuna ; Ohashi, Yasuko ; Mikami, Hirofumi ; Yamamoto, Mizuki ; Ihara, Tatsuya ; Kumazawa, Kentaro ; Sugihara, Kosuke ; Goshima, Naoki ; Watanabe, Shinya ; Senba, Kentaro. / A method of producing genetically manipulated mouse mammary gland. In: Breast cancer research : BCR. 2019 ; Vol. 21, No. 1.
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    AU - Tagaya, Hiroaki

    AU - Ishikawa, Kosuke

    AU - Hosokawa, Yoshito

    AU - Kobayashi, Shun

    AU - Ueoka, Yukino

    AU - Shimada, Mayuna

    AU - Ohashi, Yasuko

    AU - Mikami, Hirofumi

    AU - Yamamoto, Mizuki

    AU - Ihara, Tatsuya

    AU - Kumazawa, Kentaro

    AU - Sugihara, Kosuke

    AU - Goshima, Naoki

    AU - Watanabe, Shinya

    AU - Senba, Kentaro

    PY - 2019/1/5

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    N2 - BACKGROUND: To obtain a deep understanding of the mechanism by which breast cancer develops, the genes involved in tumorigenesis should be analyzed in vivo. Mouse mammary gland can regenerate completely from a mammary stem cell (MaSC), which enables us to analyze the effect of gene expression and repression on tumorigenesis in mammary gland regenerated from genetically manipulated MaSCs. Although lentiviral and retroviral systems have usually been applied for gene transduction into MaSCs, they are associated with difficulty in introducing long, repeated, or transcriptional termination sequences. There is thus a need for an easier and quicker gene delivery system. METHODS: We devised a new system for gene delivery into MaSCs using the piggyBac transposon vectors and electroporation. Compared with viral systems, this system enables easier and quicker transfection of even long, repeated, or transcriptional termination DNA sequences. We designed gene expression vectors of the transposon system, equipped with a luciferase (Luc) expression cassette for monitoring gene transduction into regenerative mammary gland in mice by in-vivo imaging. A doxycycline (Dox)-inducible system was also integrated for expressing the target gene after mammary regeneration to mimic the actual mechanism of tumorigenesis. RESULTS: With this new gene delivery system, genetically manipulated mammary glands were successfully reconstituted even though the vector size was > 200 kb and even in the presence of DNA elements such as promoters and transcription termination sequences, which are major obstacles to viral vector packaging. They differentiated correctly into both basal and luminal cells, and showed normal morphological change and milk production after pregnancy, as well as self-renewal capacity. Using the Tet-On system, gene expression can be controlled by the addition of Dox after mammary reconstitution. In a case study using polyoma-virus middle T antigen (PyMT), oncogene-induced tumorigenesis was achieved. The histological appearance of the tumor was highly similar to that of the mouse mammary tumor virus-PyMT transgenic mouse model. CONCLUSIONS: With this system, gene transduction in the mammary gland can be easily and quickly achieved, and gene expression can be controlled by Dox administration. This system for genetic manipulation could be useful for analyzing genes involved in breast cancer.

    AB - BACKGROUND: To obtain a deep understanding of the mechanism by which breast cancer develops, the genes involved in tumorigenesis should be analyzed in vivo. Mouse mammary gland can regenerate completely from a mammary stem cell (MaSC), which enables us to analyze the effect of gene expression and repression on tumorigenesis in mammary gland regenerated from genetically manipulated MaSCs. Although lentiviral and retroviral systems have usually been applied for gene transduction into MaSCs, they are associated with difficulty in introducing long, repeated, or transcriptional termination sequences. There is thus a need for an easier and quicker gene delivery system. METHODS: We devised a new system for gene delivery into MaSCs using the piggyBac transposon vectors and electroporation. Compared with viral systems, this system enables easier and quicker transfection of even long, repeated, or transcriptional termination DNA sequences. We designed gene expression vectors of the transposon system, equipped with a luciferase (Luc) expression cassette for monitoring gene transduction into regenerative mammary gland in mice by in-vivo imaging. A doxycycline (Dox)-inducible system was also integrated for expressing the target gene after mammary regeneration to mimic the actual mechanism of tumorigenesis. RESULTS: With this new gene delivery system, genetically manipulated mammary glands were successfully reconstituted even though the vector size was > 200 kb and even in the presence of DNA elements such as promoters and transcription termination sequences, which are major obstacles to viral vector packaging. They differentiated correctly into both basal and luminal cells, and showed normal morphological change and milk production after pregnancy, as well as self-renewal capacity. Using the Tet-On system, gene expression can be controlled by the addition of Dox after mammary reconstitution. In a case study using polyoma-virus middle T antigen (PyMT), oncogene-induced tumorigenesis was achieved. The histological appearance of the tumor was highly similar to that of the mouse mammary tumor virus-PyMT transgenic mouse model. CONCLUSIONS: With this system, gene transduction in the mammary gland can be easily and quickly achieved, and gene expression can be controlled by Dox administration. This system for genetic manipulation could be useful for analyzing genes involved in breast cancer.

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    KW - Electroporation

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    KW - Transgenesis

    KW - Transposon

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