Transgenic mice with a tandem duplication of the Necdin gene overexpress Necdin

Abstract

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.

Imprinting analysis by droplet digital PCR coupled with locked nucleic acid TaqMan probes

“…Allelic discrimination of transcripts with low expression and analysis of the skewness in allelic expression are difficult to perform by conventional densitometry, enzymatic digestions, or chromatographic sequencing of RT-PCR products. We recently used the ddPCR assay with LNA TaqMan probes for imprinting analysis of tissues with faint Ndn expression [28]. Here, we applied it to analyse tissue-and cell-specific Ube3a imprinting.…”