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.

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

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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 - Ohashi, Yasuko

AU - Mikami, Hirofumi

AU - Yamamoto, Mizuki

AU - Ihara, Tatsuya

AU - Kumazawa, Kentaro

AU - Sugihara, Kosuke

AU - Goshima, Naoki

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AU - Senba, Kentaro

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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.

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