Reference - Detail
|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, Semba K.
|A method of producing genetically manipulated mouse mammary gland.
|Breast Cancer Res
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.
|Animals Cell Differentiation / genetics* Cell Line DNA Transposable Elements / genetics DNA-Binding Proteins / genetics Doxycycline / administration & dosage Female Fibroblasts Gene Expression Regulation / drug effects Genes, Reporter Genetic Engineering / methods* Genetic Vectors / genetics Green Fluorescent Proteins / genetics Mammary Glands, Animal / cytology Mammary Glands, Animal / physiology* Mammary Glands, Animal / transplantation Mammary Neoplasms, Experimental / genetics* Mammary Neoplasms, Experimental / pathology Mice Mice, Inbred C57BL Mice, Knockout Primary Cell Culture / methods Stem Cells / physiology* Transfection / methods
|B6N Mouse BAC clone B6Ng01-263 (RDB07573)
|Human and Animal Cells