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Protocol: Adeno-Associated Virus-Mediated Gene Transfer in Ex Vivo Cultured Embryonic Mammary Gland

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...

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Autores principales: Lan, Qiang, Mikkola, Marja L.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Springer US 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7960627/
https://www.ncbi.nlm.nih.gov/pubmed/33009602
http://dx.doi.org/10.1007/s10911-020-09461-4
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author Lan, Qiang
Mikkola, Marja L.
author_facet Lan, Qiang
Mikkola, Marja L.
author_sort Lan, Qiang
collection PubMed
description 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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s10911-020-09461-4) contains supplementary material, which is available to authorized users.
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spelling pubmed-79606272021-04-01 Protocol: Adeno-Associated Virus-Mediated Gene Transfer in Ex Vivo Cultured Embryonic Mammary Gland Lan, Qiang Mikkola, Marja L. J Mammary Gland Biol Neoplasia Article 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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s10911-020-09461-4) contains supplementary material, which is available to authorized users. Springer US 2020-10-02 2020 /pmc/articles/PMC7960627/ /pubmed/33009602 http://dx.doi.org/10.1007/s10911-020-09461-4 Text en © The Author(s) 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
spellingShingle Article
Lan, Qiang
Mikkola, Marja L.
Protocol: Adeno-Associated Virus-Mediated Gene Transfer in Ex Vivo Cultured Embryonic Mammary Gland
title Protocol: Adeno-Associated Virus-Mediated Gene Transfer in Ex Vivo Cultured Embryonic Mammary Gland
title_full Protocol: Adeno-Associated Virus-Mediated Gene Transfer in Ex Vivo Cultured Embryonic Mammary Gland
title_fullStr Protocol: Adeno-Associated Virus-Mediated Gene Transfer in Ex Vivo Cultured Embryonic Mammary Gland
title_full_unstemmed Protocol: Adeno-Associated Virus-Mediated Gene Transfer in Ex Vivo Cultured Embryonic Mammary Gland
title_short Protocol: Adeno-Associated Virus-Mediated Gene Transfer in Ex Vivo Cultured Embryonic Mammary Gland
title_sort protocol: adeno-associated virus-mediated gene transfer in ex vivo cultured embryonic mammary gland
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7960627/
https://www.ncbi.nlm.nih.gov/pubmed/33009602
http://dx.doi.org/10.1007/s10911-020-09461-4
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