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

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.

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

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.

Imprinting analysis of the mouse chromosome 7C region in DNMT1-null embryos.

The mouse chromosome 7C, orthologous to the human 15q11-q13 has an imprinted domain, where most of the genes are expressed only from the paternal allele. The imprinted domain contains paternally expressed genes, Snurf/Snrpn, Ndn, Magel2, Mkrn3, and Frat3, C/D-box small nucleolar RNAs (snoRNAs), and the maternally expressed gene, Ube3a. Imprinted expression in this large (approximately 3-4 Mb) domain is coordinated by a bipartite cis-acting imprinting center (IC), located upstream of the Snurf/Snrpn gene. The molecular mechanism how IC regulates gene expression of the whole domain remains partially understood. Here we analyzed the relationship between imprinted gene expression and DNA methylation in the mouse chromosome 7C using DNA methyltransferase 1 (DNMT1)-null mutant embryos carrying Dnmt1(ps) alleles, which show global loss of DNA methylation and embryonic lethality. In the DNMT1-null embryos at embryonic day 9.5, the paternally expressed genes were biallelically expressed. Bisulfite DNA methylation analysis revealed loss of methylation on the maternal allele in the promoter regions of the genes. These results demonstrate that DNMT1 is necessary for monoallelic expression of the imprinted genes in the chromosome 7C domain, suggesting that DNA methylation in the secondary differentially methylated regions (DMRs), which are acquired during development serves primarily to control the imprinted expression from the maternal allele in the mouse chromosome 7C.