Cell and tissue dynamics research program, Institute of Biotechnology, Helsinki Institute of Life Science (HiLIFE), University of Helsinki, Helsinki, Finland.
J Mammary Gland Biol Neoplasia. 2020 Dec;25(4):409-416. doi: 10.1007/s10911-020-09461-4. Epub 2020 Oct 2.
Branching morphogenesis of the murine mammary gland starts during late embryogenesis. It is regulated by the signals emanating both from the epithelium and the mesenchyme, yet the molecular mechanisms regulating this process remain poorly understood. We have previously developed a unique whole organ culture technique for embryonic mammary glands, which provides a powerful tool to monitor and manipulate branching morphogenesis ex vivo. Nowadays, RNA sequencing and other transcriptional profiling techniques provide robust methods to identify components of gene regulatory networks driving branching morphogenesis. However, validation of the candidate genes still mainly depends on the use of the transgenic mouse models, especially in mammary gland studies. By comparing different serotypes of recombinant adeno-associated virus (rAAVs), we found out that rAAVs provide sufficient efficiency for gene transfer with different tissue preferences depending on the serotypes of the virus. AAV-2 and AAV-8 preferentially target epithelial and mesenchymal compartments, respectively, while AAV-9 infects both tissues. Here, we describe a protocol for AAV-mediated gene transfer in ex vivo cultured murine embryonic mammary gland facilitating gene function studies on mammary gland branching morphogenesis.
分支形态发生的小鼠乳腺开始于胚胎后期。它是由上皮和间充质发出的信号调节的，但调节这一过程的分子机制仍知之甚少。我们之前开发了一种独特的胚胎乳腺整体器官培养技术，为体外监测和操作分支形态发生提供了有力工具。如今，RNA 测序和其他转录谱分析技术为鉴定驱动分支形态发生的基因调控网络的组成部分提供了强大的方法。然而，候选基因的验证仍然主要依赖于使用转基因小鼠模型，特别是在乳腺研究中。通过比较不同血清型的重组腺相关病毒 (rAAV)，我们发现 rAAV 提供了足够的基因转移效率，不同血清型的病毒具有不同的组织偏好。AAV-2 和 AAV-8 分别优先靶向上皮和间充质区室，而 AAV-9 感染这两种组织。在这里，我们描述了一种在体外培养的小鼠胚胎乳腺中进行 AAV 介导的基因转移的方案，该方案有助于研究乳腺分支形态发生中的基因功能。
