類DNA甲基化轉移酶-3L與 Trichostatin A處理於複製小鼠體細胞核移置之再程序化過程探討

Abstract

體細胞核移置技術係利用卵母細胞成功地再程序化體細胞核以產製複製動物。此外目前體細胞核移置不只是一項動物複製科技，已逐漸成為基礎學理、再生醫學及農業應用之重要研究工具。然而，複製過程中體細胞核再程序化不完全而導致後生遺傳修飾異常，故整體效率仍然很低。 DNA甲基化與組蛋白乙醯化（histone acetylation）皆為重要的後生遺傳修飾作用，對早期胚發育及配子生成時具有深遠影響；發育過程中重新甲基化（de novo methylation）之過程主要由DNA甲基化轉移酶3家族（DNA methyltransferase 3）負責，包含Dnmt3a, Dnmt3b與Dnmt3L。其中Dnmt3L主要功能為調控Dnmt3a及Dnmt3b以進行重新甲基化，近來發現Dnmt3L可與組蛋白去乙醯化酶1（Histone deacetylase 1, HDAC1）結合後對組蛋白進行去乙醯化，進而調控基因之表現。因此，若將Dnmt3L 剔除後能否降低複製胚中過度甲基化（hypermethylation），抑或減少組蛋白去乙醯化，進而提升與胚發育相關基因之表現，後而改善體細胞核移置效率。另一方面，Trichostatin A（TSA）為HDACs的抑制物，在先前研究中顯示其可提高複製胚中乙醯化程度，而顯著提昇體細胞核移置之成功率。吾等推測將供核細胞中之Dnmt3L剔除後並配合TSA處理，可能具有改善體細胞核再程序化之可能性。因此，本試驗研究旨在利用體細胞核移置技術探討Dnmt3L於再程序化過程中所扮演之角色，另一則是以Dnmt3L-KO(Dnmt3L-knockout, Dnmt3L-KO)小鼠之胎兒纖維母細胞進行核移置後配合TSA處理，探討其對複製小鼠胚體外發育潛能之影響。 本實驗利用不同來源之供核細胞，分別來自野生型(wild type, Wt)以及Dnmt3L-KO小鼠之胎兒纖維母細胞進行核移置，並依照試驗設計添加10 nm之TSA處理，比較複製胚體外發育至囊胚之效率。結果顯示，經TSA處理後可顯著提升野生型(32% vs. 4%)以及Dnmt3L-KO(63% vs. 8%)兩組供核細胞之囊胚發育率。除此之外，利用免疫螢光染色法偵測複製囊胚中octamer-4（Oct4）以及caudal type homeobox transcription factor 2（Cdx2）之基因表現，依照Oct4及Cdx2分布的狀況，將囊胚區分為Grade 1到Grade 4四個等級，並且配合囊胚中細胞總數作為判斷囊胚之品質之依據。結果顯示，Dnmt3L-KO小鼠之胎兒纖維母細胞配合TSA處理之複製小鼠囊胚，具有較多的細胞數，並且正常分布Oct4及Cdx2的囊胚比例（Grade 1與Grade 2）亦高於其他處理組。 綜合上述，Dnmt3L-KO與TSA可能存在協同作用，不僅可顯著提升囊胚發育率，對於囊胚之品質也有所改善，然而Dnmt3L-KO與TSA之交互作用關係仍需進一步研究結果釐清。

It has been more than a decade since “Dolly” the sheep was produced via somatic cell nuclear transfer (SCNT). Successful reprogramming of the differentiated cell by SCNT has been reported for more than 15 mammalian species that resulted in the live clones. Therefore, SCNT technology has been recognized as a powerful tool to insight into various fundamental studies in cellular, developmental, and molecular biology, as well as hold great promise in applications of agriculture and regenerative medicine. However, the cloning efficiency has remained extremely low, and was believed due to incomplete reprogramming of the somatic nuclei that contributed by insufficient epigenetic modifications. DNA methylation plays an important epigenetic modification of gene expression in early development. It has been demonstrated that DNA methyltransferase (Dnmt) activity, including Dnmt3L, interacted with histone deacetylase (HDAC), was involved in histone deacetylation, chromatin remodeling and transcription repression. On the other hand, Trichostatin A (TSA), a histone deacetylase inhibitor (HADCi) has a function of increase histone acetylation and DNA demethylation, has been demonstrated improve the efficiency of nuclear cloning in numerous of species. If knock out Dnmt3L (Dnmt3L-KO), will it affect histone acetylation or methylation of reconstructed embryos following SCNT? Any beneficial effect during reprogramming process when combine Dnmt3L-KO nuclei with TSA treatment after SCNT? Therefore, the object of this study is to determine: 1) the role of Dnmt3L during nuclear reprogramming by SCNT, and 2) the reprogramming potential of Dnmt3L-KO fibroblast cells by nuclear transfer following TSA treatment. We compare cloning efficiency by using mouse embryonic fibroblasts derived from wild type or Dnmt3L-KO mice as donor cells for SCNT and reconstructed embryos were treated with 10 nM TSA for10 h immediately after activation. We found that cloning efficiency from both wild type and Dnmt3L-KO groups were significantly increased following TSA treatment. The development rates of cloned blastocysts compared with those observed in non-treated control groups were: 32% vs. 4% in wild type, 63% vs. 8% in D3L-KO- derived embryos. In addition, we use antibodies to detect the distribution of Oct-4/Cdx2 and divided them to Grade 1 (good) to Grade 4 (bad) together with total cell number to classify quality of cloned embryos. Combination of Dnmt3L and TSA increased not only in good quality of Grade 1 embryos but also in total cell number of cloned blastocyst. Our data indicate that TSA treatment significantly increased the developmental potential of cloned embryos and there seems a synergistic effect between Dnmt3L-KO and TSA treatment during nuclear reprogramming by SCNT.