Bidirectional approach for elucidation of genomic imprinting mechanism

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Genomic imprinting is an epigenetic phenomenon that is responsible for parent-of-origin specific expression of genes. A disruption of imprinting at 11p 15.5 develops Beckwith-Wiedemann syndrome (BWS) and tumors. To clarify a molecular mechanism of genomic imprinting, we studied following issues.1. Analysis of Japanese cases of BWS revealed a significantly lower frequency of H19-DMR hypermethylation and a higher frequency of chromosome abnormality than in North American and European patients. These results suggest that susceptibility to epigenetic and genetic alterations differs between the two groups.2. Truncation cells in which short transcripts, 0.2 kb, 1.1 kb, and 6.6 kb, of LIT1were expressed were generated using a mouse hybrid cell containing human paternal chromosome 11. qRT-PCR revealed that expression of an imprinted KvLQT1 gene was drastically elevated in these cells. The result suggested that a non-coding transcript of LIT1 gene regulates expression of imprinted genes.3. To identify a novel regulator for genomic imprinting, we tried to generate specific cells, which are able to be detected an imprinting disruption by neomycin-resistance. We constructed a knock-in vector in which IRES-neo cassette were inserted into downstream of an imprinted Kip2 gene. The vector was transfected into mouse ES cells ; however, no recombinant clone was obtained to date. Thus, we changed a construction of the vector, and we are now screening of ES cells. After obtained recombinant clones, we will generate Kip2-IRES-neo knock-in mice and establish mouse embryonic fibroblast.