Cloning and analysis of a candidate imprinted gene identified by mouse cDNA subtraction hybridization

Abstract

Mouse parthenogenones are expected not to express maternally imprinted genes since their genomes are derived exclusively from female pronuclei. On the basis of this assumption, a pool of cDNAs from normal mouse embryos was subtracted by that of partheno- genetic mouse embryos to obtain cDNA fragments containing sequences of maternally imprinted genes. Obtained were seven cDNA candidate fragments, among which a 200 bp fragment exhibited differential hybridization between normal and partheno- genetic cDNA pools. In this study, a mouse testis cDNA library was constructed and screened for cDNAs containing DNA sequences of the 200 bp fragment. Two cDNA clones, Nep1-1 and Nep1-2 were isolated and their insert DNAs were sequenced. A cDNA contig of 760 bp length was assembled from previously reported EST sequences as well as from sequences of Nep1-1 and Nep1-2. To investigate the genomic status of this gene, Southern blot analysis was carried out. The result indicated that the Nep1 gene is present as a single copy in the mouse genome. It was also shown that sites of differential cytosine methylations exist near the locus of Nep1 gene. The possibility of imprinting of the Nep1 gene was supported by this result since differential expression of parental alleles generally involves differential DNA methylation. Expression of the Nep1 gene in various tissues was assessed by a Northern hybridization experiment using the 200 bp fragment as a probe. Surprisingly, the Nep1 gene was expressed predomintly in testis, while expressed rarely in other tissues including 10.5 dpc embryos from which the probe cDNA itself was derived. Therefore, it was postulated that Nep1 gene is tissue-specifically expressed from the paternal allele.