A method of producing genetically manipulated mouse mammary gland

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.Electronic supplementary materialThe online version of this article (10.1186/s13058-018-1086-8) contains supplementary material, which is available to authorized users.

piggyBac-Based Non-Viral In Vivo Gene Delivery Useful for Production of Genetically Modified Animals and Organs

“…Mouse mammary glands can regenerate completely by the use of mammary stem cells (MaSCs). To examine the mechanism by which breast cancer develops, Tagaya et al [68] constructed bacterial artificial chromosome (BAC)-based PB transposons with a vector size of >200 kb to transfect MaSCs via in vitro EP. They transplanted the transfected MaSCs into cleared fat pads of the inguinal mammary glands (from which the endogenous epithelium had been removed) of immune-deficient mice.…”

“…They observed correct differentiation of the transplanted cells into both basal and luminal cells, as well as milk production after pregnancy. Tagaya et al [68] also demonstrated that oncogene-induced tumorigenesis is possible when MaSCs transfected with PB transposons, which carry the polyoma-virus middle T antigen gene, are transplanted into mammary glands.…”

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“…However, this engineering approach is not practical for highthroughput and larger-scale gene evaluation. Recently, we reported a transposon-based genetic manipulation system in the mammary gland that can be applied to oncogene screening, lineage tracing, and marker gene screening [1].…”

Genetic manipulation of the mammary gland and potential applications

“…However, this engineering approach is not practical for high-throughput and larger-scale gene evaluation. Recently, we reported a transposon-based genetic manipulation system in the mammary gland that can be applied to oncogene screening, lineage tracing, and marker gene screening [1].…”

“…However, there are intrinsic limitations in introducing long or complex sequences that include multiple promoters or transcription termination sequences owing to their viral packaging mechanism. To address this problem, we recently established another method using the piggyBac transposon system combined with electroporation [1]. The transposon system has almost no restrictions on sequence type or cargo length (> 200 kb BAC) [7], so that the design of complex and long DNA elements, including those for overexpression, knockout, or knockdown experiments, are almost limitless.…”