Necdin (Ndn) transgenic (Tg) mice were generated with a bacterial artificial chromosome (BAC) clone. Droplet digital PCR (ddPCR) and inverse PCR methods revealed that the transgene consisted of four fragments with a total length of 171 kb. Two of these fragments were tandem tail-to-tail duplicates of 77 kb and 37 kb that both contained a Ndn gene. The transgene was inserted in chromosome 15qD1. Ndn is a paternally expressed imprinted gene; however, the total expression level of Ndn in hemizygous Tg mice was approximately twofold higher than that in wild-type mice. ddPCR assays with locked nucleic acid (LNA) TaqMan probes revealed that transgenic Ndn expression was almost equal to endogenous Ndn expression, despite there being two copies of the Ndn gene in the transgene, indicating an interaction between the transcriptional regulation of endogenous Ndn and the transgene. ddPCR assays with LNA TaqMan probes were also applied for imprinting analysis to confirm exclusive paternal expression in tissues with low Ndn expression. This is the first report of a Tg mouse with a tandem duplication of a Ndn transgene and Ndn overexpression, which will be useful for the in vivo study of Ndn overexpression and for rescue experiments of the neonatal lethality seen in the Ndn knockout mouse.

中文翻译：

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Necdin基因串联重复的转基因小鼠过表达Necdin。

用细菌人工染色体（BAC）克隆产生了Necdin（Ndn）转基因（Tg）小鼠。液滴数字PCR（ddPCR）和反向PCR方法表明，转基因由四个片段组成，总长度为171 kb。这些片段中的两个片段是77 kb和37 kb的串联尾到尾重复序列，均包含Ndn基因。转基因被插入染色体15qD1。Ndn是一个父系表达的印迹基因。然而，半合子Tg小鼠中Ndn的总表达水平比野生型小鼠高约两倍。使用锁定核酸（LNA）TaqMan探针的ddPCR分析表明，尽管转基因中有两个Ndn基因拷贝，但转基因Ndn表达几乎等于内源性Ndn表达，表明内源性Ndn的转录调控与转基因之间存在相互作用。使用LNA TaqMan探针的ddPCR分析也用于印记分析，以确认低Ndn表达的组织中只有父本表达。这是Tg小鼠串联Ndn转基因和Ndn过表达的双重报道，这对Ndn过表达的体内研究以及对Ndn基因敲除小鼠的新生儿致死性的抢救实验很有用。