Bidirectional approach for elucidation of genomic imprinting mechanism

基因组印迹是一种表观遗传现象，它负责基因的亲本特异性表达。位于11p15.5的印迹中断会发展为Beckwith-Wiedemann综合征(BWS)和肿瘤。为了阐明基因组印迹的分子机制，我们研究了以下几个问题。对日本BWS病例的分析显示，与北美和欧洲患者相比，H19-DMR高甲基化频率显著降低，而染色体异常频率更高。这些结果表明，对表观遗传和遗传改变的易感性在两组之间是不同的。用含人父系11号染色体的小鼠杂交细胞获得表达LIT1短转录本(0.2kb、1.1kb和6.6kb)的截短细胞。QRT-PCR结果显示印记KvLQT1基因在这些细胞中的表达水平显著升高。这一结果提示LIT1基因的非编码转录本调控印记基因的表达。为了确定一种新的基因组印迹调控因子，我们试图产生特定的细胞，这些细胞能够检测到新霉素抗性造成的印迹破坏。我们构建了一个敲入载体，将IRES-neo盒插入印记Kip2基因的下游。将该载体导入小鼠ES细胞，但至今未获得重组克隆。因此，我们改变了载体的结构，现在我们正在筛选ES细胞。获得重组克隆后，我们将产生Kip2-IRES-neo敲入小鼠，并建立小鼠胚胎成纤维细胞。

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.