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.

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ALK gene rearrangement is observed in approximately 4% of patients with non-small cell lung cancer. These individuals benefit clinically from a range of approved ALK-TKIs; however, using many ALK-TKIs in a row will always result in resistant compound mutations in the kinase domain. Therefore, next-generation ALK-TKIs which are potent to these resistant mutations are still under development. In this context, preclinical prediction of resistant mutations generated by developing ALK-TKIs will provide useful information about the effective sequential treatment of ALK-TKIs. In this study, we developed a simple error-prone PCR-based mutation prediction system, and as a model study, we predicted resistant mutations against upcoming ALK-TKI, repotrectinib, and ensartinib. According to the predictive mutation patterns, repotrectinib and ensartinib may be used as second-line therapeutic options following the first-line alectinib treatment.