A method of producing genetically manipulated mouse mammary gland

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

doi:10.1186/s13058-018-1086-8

Abstract

BackgroundTo 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.MethodsWe 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.ResultsWith 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.ConclusionsWith 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.

Highlights

To obtain a deep understanding of the mechanism by which breast cancer develops, the genes involved in tumorigenesis should be analyzed in vivo
When a tumorigenic event such as gene amplification occurs [1], Mammary epithelial cell (MEC) acquire the potential to proliferate in the mammary duct and develop into ductal carcinoma in situ (DCIS) [2], which progresses to malignant carcinoma such as invasive breast carcinoma and metastasizes into other organs through mesenchymal tissue
DNA elements between the inverted terminal repeat (ITR) sequences of transposon can be integrated into the host genome via a cut-and-paste mechanism by the expression of transposase

Summary

Introduction

To obtain a deep understanding of the mechanism by which breast cancer develops, the genes involved in tumorigenesis should be analyzed in vivo. For in-vivo analysis of the genes involved in these processes, some genetically manipulated mouse models have been established, such as TP53−/−, mouse mammary tumor virus (MMTV)-neu, and MMTV-polyoma-virus middle T antigen (PyMT) mice. These models have boosted our understanding of the mechanisms by which mammary tumors develop and metastasize in vivo [3,4,5]. Transgenic or knockout mouse models are useful for understanding the tumorigenic activity of those genes, but have not been practical for analyzing multiple candidate genes. We have to establish a novel alternative method for preparing genetically engineered mice to improve the efficiency for analysis of multiple genes

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Conclusion