Transgenic mice with a tandem duplication of the Necdin gene overexpress Necdin
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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.
This work was supported by JSPS KAKENHI Grant Number 25461555 (T.K.). We thank Katsunori Hironaka (Bio-Rad Laboratories, Japan) for helpful suggestions regarding ddPCR analysis. We thank Jeremy Allen, PhD, from Edanz Group (http://www.edanzediting.com/ac) for editing a draft of this manuscript.
SH and AU generated and maintained the mouse strains, AN performed RT-PCR, qPCR, and ddPCR assays, MM performed methylation assays, TK designed the study, analyzed results, and drafted the manuscript. All authors reviewed the manuscript.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
All procedures involving animals were performed in accordance with the ethical standards of the Nagasaki University Institutional Animal Care and Use Committee.
- Rieusset A, Schaller F, Unmehopa U, Matarazzo V, Watrin F, Linke M, Georges B, Bischof J, Dijkstra F, Bloemsma M, Corby S, Michel FJ, Wevrick R, Zechner U, Swaab D, Dudley K, Bezin L, Muscatelli F (2013) Stochastic loss of silencing of the imprinted Ndn/NDN allele, in a mouse model and humans with prader-willi syndrome, has functional consequences. PLoS Genet 9(9):e1003752. https://doi.org/10.1371/journal.pgen.1003752 CrossRefPubMedPubMedCentralGoogle Scholar
- Watrin F, Roeckel N, Lacroix L, Mignon C, Mattei MG et al (1997) The mouse Necdin gene is expressed from the paternal allele only and lies in the 7C region of the mouse chromosome 7, a region of conserved synteny to the human Prader-Willi syndrome region. Eur J Hum Genet 5:324–332PubMedGoogle Scholar