Tosh Justin L, Rickman Matthew, Rhymes Ellie, Norona Frances E, Clayton Emma, Mucke Lennart, Isaacs Adrian M, Fisher Elizabeth M C, Wiseman Frances K
Department of Neurodegenerative Disease, Institute of Neurology, University College London, London, WC1N 3BG, UK.
Gladstone Institute of Neurological Disease and University of California, San Francisco, CA, 4158, USA.
Wellcome Open Res. 2018 Oct 10;2:84. doi: 10.12688/wellcomeopenres.12237.2. eCollection 2017.
Transgenic animal models are a widely used and powerful tool to investigate human disease and develop therapeutic interventions. Making a transgenic mouse involves random integration of exogenous DNA into the host genome that can have the effect of disrupting endogenous gene expression. The J20 mouse model of Alzheimer's disease (AD) is a transgenic overexpresser of human APP with familial AD mutations and has been extensively utilised in preclinical studies and our aim was to determine the genomic location of the J20 transgene insertion. We used a combination of breeding strategy and Targeted Locus Amplification with deep sequencing to identify the insertion site of the J20 transgene array. To assess RNA and protein expression of we used qRT-PCR and Western Blotting. We demonstrate that the J20 transgene construct has inserted within the genetic locus of endogenous mouse gene on chromosome 16 in an array disrupting expression of mRNA from this gene in adult hippocampal tissue. Preliminary data suggests that ZBTB20 protein levels remain unchanged in this tissue, however further study is necessary. We note that the endogenous mouse gene also lies on chromosome 16, although 42 Mb from the locus. These data will be useful for future studies utilising this popular model of AD, particularly those investigating gene interactions between the J20 transgene and other genes present on Mmu16 in the mouse.
转基因动物模型是研究人类疾病和开发治疗干预措施的一种广泛使用且强大的工具。制作转基因小鼠涉及将外源DNA随机整合到宿主基因组中，这可能会破坏内源基因的表达。阿尔茨海默病（AD）的J20小鼠模型是具有家族性AD突变的人类APP转基因过表达模型，已在临床前研究中广泛使用，我们的目的是确定J20转基因插入的基因组位置。我们结合使用育种策略和靶向基因座扩增及深度测序来鉴定J20转基因阵列的插入位点。为了评估RNA和蛋白质表达，我们使用了qRT-PCR和蛋白质免疫印迹法。我们证明J20转基因构建体已插入到16号染色体上内源性小鼠基因的基因座内，形成一个阵列，破坏了该基因在成年海马组织中的mRNA表达。初步数据表明该组织中ZBTB20蛋白水平保持不变，然而还需要进一步研究。我们注意到内源性小鼠基因也位于16号染色体上，尽管与该基因座相距42 Mb。这些数据将有助于未来利用这种流行的AD模型进行的研究，特别是那些研究J20转基因与小鼠Mmu16上存在的其他基因之间基因相互作用的研究。
