Protocol: Adeno-Associated Virus-Mediated Gene Transfer in Ex Vivo Cultured Embryonic Mammary Gland
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Protocol: Adeno-Associated Virus-Mediated Gene Transfer in Ex Vivo Cultured Embryonic Mammary Gland Qiang Lan 1
&
Marja L. Mikkola 1
Received: 9 July 2020 / Accepted: 24 September 2020 # The Author(s) 2020
Abstract 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. Keywords Organ culture . Ex vivo . Explant culture . Adeno-associated virus
Introduction The embryonic development of the mammary gland in mouse starts at embryonic day (E) 10 with the formation of milk lines, which are two discontinued stripes of ventral lateral surface ectoderm between the fore and hind limb [1–3]. One day later, 5 pairs of symmetrically positioned mammary rudiments named placodes have formed, presumably by cell migration [4, 5]. By E13, placodes have extended into the underlying mesenchyme to form buds and are now surrounded by a condensed, mammary-specific mesenchyme [6, 7]. Starting from E15-E16, depending on the strain of mouse, Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10911-020-09461-4) contains supplementary material, which is available to authorized users. * Qiang Lan [email protected] * Marja L. Mikkola [email protected] 1
Cell and tissue dynamics research program, Institute of Biotechnology, Helsinki Institute of Life Science (HiLIFE), University of Helsinki, Helsinki, Finland
the mammary bud starts sprouting and invades into the underlying fat pad, the precursor of adult mammary gland stroma. By E18.5 (just before birth), the mammary gland forms a small ductal tree with about 10–15 branches. After birth, mammary gland development is slow until the onset of the puberty. The interaction between mesenchymal and epithelial cells is essential for mammary gland morphogenesis at all developmental stages. Due to the technical difficu
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