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Application of droplet digital PCR in the analysis of genome integration and organization of the transgene in BAC transgenic mice


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Title: Application of droplet digital PCR in the analysis of genome integration and organization of the transgene in BAC transgenic mice
Authors: Nakagaki, Ayumi / Urakawa, Asuka / Hirano, Shiori / Anami, Takeru / Kishino, Tatsuya
Issue Date: 27-Apr-2018
Publisher: Nature Publishing Group
Citation: Scientific Reports, 8, 6638; 2018
Abstract: Transgenic (Tg) mice containing bacterial artificial chromosome (BAC) DNA are widely used for gene expression analysis and gene therapy models because BAC transgenes provide gene expression at physiological levels with the same developmental timing as endogenous genes. To ensure correct interpretation of transgene functions, investigation of the genomic organisation and integration of the BAC transgene is required. Here, we describe a reliable method based on droplet digital PCR (ddPCR) and inverse PCR to estimate copy number, genomic organisation and insertion sites of BAC transgenes in the mouse genome. We generated BAC Tg mice containing fragments of BAC clone RP23-59P20. ddPCR and iPCR analysis showed that the transgene consisted of five fragments of the BAC clone containing the Mkrn3 gene region, and that the transgene was inserted into Bckdhb, homozygous deletion of which causes the maple syrup urine disease phenotype. The ddPCR method described here should prove useful for analysis of genomic organisation and integration of BAC transgenes.
URI: http://hdl.handle.net/10069/38216
DOI: 10.1038/s41598-018-25001-x
Rights: © 2018 The Author(s). / 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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/licenses/by/4.0/.
Type: Journal Article
Text Version: publisher
Appears in Collections:Articles in academic journal

Citable URI : http://hdl.handle.net/10069/38216

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