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

Ayumi Nakagaki,Tatsuya Kishino,Shiori Hirano,Asuka Urakawa,Takeru Anami

doi:10.1038/s41598-018-25001-x

Ayumi Nakagaki, Tatsuya Kishino + Show 3 more

Open Access

https://doi.org/10.1038/s41598-018-25001-x

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Journal: Scientific Reports	Publication Date: Apr 27, 2018
Citations: 2	License type: open-access

Affiliation: Nagasaki University, Nagasaki Medical Center

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.

Highlights

Bacterial artificial chromosomes (BAC) are useful resources for long-range analysis of genomic organisation and gene function
This study introduces the applicability of droplet digital PCR (ddPCR) for quantitative detection of copy numbers of transgenes, leading to identification of the integration sites of transgenes by inverse PCR (iPCR) in bacterial artificial chromosome (BAC) transgenic mice
The genomic organization of the transgene was analysed using the ddPCR and iPCR method, revealing a complicated genomic organisation whereby the transgene was inserted into the branched-chain keto acid dehydrogenase E1 beta polypeptide gene (Bckdhb), homozygous mutations in which result in maple syrup urine disease (MSUD)

Summary

Introduction

Bacterial artificial chromosomes (BAC) are useful resources for long-range analysis of genomic organisation and gene function. We developed a simple method to investigate the genomic organisation and integration sites of transgenes using a combination of droplet digital PCR (ddPCR) and inverse PCR (iPCR). In mice, the loss of all paternally-expressed genes in the critical region results in severe failure to thrive in early life, yielding neonatal lethality[8,9,10]. To rescue these mice, exogenous gene expression in the PWS critical region is needed, but has not yet been achieved. The genomic organization of the transgene was analysed using the ddPCR and iPCR method, revealing a complicated genomic organisation whereby the transgene was inserted into the branched-chain keto acid dehydrogenase E1 beta polypeptide gene (Bckdhb), homozygous mutations in which result in maple syrup urine disease (MSUD)

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Results

Conclusion