核移植胚胎干細(xì)胞的印跡基因甲基化研究

核移植來源的胚胎干細(xì)胞(NTES  cells)在以干細(xì)胞為基礎(chǔ)的細(xì)胞治療中扮演著非常重要的角色，得到全能性良好且表觀遺傳修飾正常的核移植胚胎干細(xì)胞是解決治療性克隆安全問題的重要前提。DNA甲基化修飾在基因表達(dá)和印跡基因的表達(dá)中起非常重要的作用，兩步法克隆可能存在的不完全重編程問題很可能存在于印跡基因的甲基化水平上，并可能遺傳到子代細(xì)胞中。因此，研究核移植胚胎干細(xì)胞的印跡基因甲基化狀態(tài)及其與全能性的關(guān)系對(duì)于干細(xì)胞依賴的細(xì)胞治療的臨床應(yīng)用具有重要理論和實(shí)際上的指導(dǎo)意義。

在本研究中，作者采用了具有不同遺傳背景(C57/DBA,  C57/129)的小鼠體細(xì)胞核移植胚胎干細(xì)胞和正常來源的胚胎干細(xì)胞作為研究對(duì)象，首先用四倍體囊胚重建的方法鑒定了六株胚胎干細(xì)胞的全能性。然后利用Bisulfite  Genomic  Sequencing并結(jié)合COBRA方法，對(duì)于六株干細(xì)胞系的三個(gè)印跡基因(H19,  Peg1,  Peg3)進(jìn)行分析。研究發(fā)現(xiàn)在長時(shí)間體外培養(yǎng)(p20)的四株核移植胚胎干細(xì)胞中均存在著不同程度的甲基化異�，F(xiàn)象。其中H19表現(xiàn)出了高度動(dòng)態(tài)的甲基化模式，而Peg3則具有相對(duì)穩(wěn)定的甲基化模式。雖然核移植來源的胚胎干細(xì)胞具有很好的全能性，但是潛在的印跡異常將成為其在細(xì)胞治療中的潛在危險(xiǎn)，篩選具有正常表觀遺傳修飾的核移植胚胎干細(xì)胞系對(duì)于干細(xì)胞的臨床應(yīng)用將是不可或缺得。

常港(中科院動(dòng)物研究所聯(lián)合培養(yǎng)博士生)為本文的第一作者；參與此項(xiàng)工作的還有，劉勝(技術(shù)員)，王鳳超博士，張郁(技術(shù)員)，寇朝輝(技術(shù)員)；北京生命科學(xué)研究所的高紹榮博士和中科院動(dòng)物研究所的陳大元研究員是本文的共同通訊作者。

此項(xiàng)研究為科技部863和北京市科委資助課題，在北京生命科學(xué)研究所高紹榮實(shí)驗(yàn)室完成。

原始出處：

Genomics, In Press, Corrected Proof, Available online 13 November 2008

Differential methylation status of imprinted genes in nuclear transfer derived ES (NT-ES) cells

Gang Chang, Sheng Liu, Fengchao Wang, Yu Zhang, Zhaohui Kou, Dayuan Chen, Shaorong Gao

Compared with fertilized embryo derived ES (F-ES) cells, somatic cell nuclear transfer (SCNT) produced ES (NT-ES) cells were proposed appropriate for cell transplantation based therapies. Although previous studies indicated that NT-ES cells and F-ES cells were transcriptionally and functionally indistinguishable, characterization of DNA methylation patterns of imprinted genes in NT-ES cells is lacking. Here, we show that DNA methylation patterns in the differentially methylated region (DMR) of paternally imprinted gene, H19, displayed distinct abnormalities in certain NT-ES and F-ES cell lines after long-term culture in vitro. DNA methylation profiles of H19 appeared very dynamic in most ES cell lines examined, either hypermethylation or hypomethylation could be observed in specific ES cell lines. In contrast to H19, maternally imprinted genes, Mest and Peg3, showed relatively stable methylation patterns in ES cells, especially Peg3, which displayed better capability in enduring long-term culture in vitro. Our results indicate that abnormal methylation profiles of certain imprinted genes could be observed in both NT-ES and F-ES cell lines after long-term culture in vitro although these cell lines were proved to be pluripotent with germline transmission competent. Stringent screening of epigenetically normal NT-ES cells might be potentially necessary for further therapeutic application of these cells.