Role of Gadd45 in pluripotency and murine embryogenesis

众所周知，动物克隆过程中体细胞核的重编程需要多能性基因（如nanog或oct 4）的广泛DNA去甲基化，这些基因在体细胞中通过DNA甲基化沉默。然而，人们对DNA去甲基化的分子机制知之甚少。我们最近发现Gadd 45 a是非洲爪蟾卵母细胞中oct 4活性DNA去甲基化的关键调节因子，它通过促进DNA修复发挥作用。在这里，我们建议研究Gadd 45介导的DNA去甲基化在小鼠胚胎干细胞（ES）和小鼠胚胎的生物学作用。虽然先前已经产生了单独的Gadd 45 a、-B和-g敲除小鼠，但这些小鼠都是存活的和可育的，可能是由于功能冗余。为了避免Gadd 45基因的冗余，我们已经开发了显性阴性（dn）Gadd 45。使用这个工具，我们将通过在ES细胞中过表达dnGadd 45（功能丧失）以及野生型Gadd 45（功能获得）来研究Gadd 45在多能性中的作用。与此同时，我们将使用dnGadd 45以及野生型Gadd 45产生和表征转基因小鼠，并重新审视该基因在早期小鼠胚胎发生中的作用。

It is well established that reprogramming of somatic cell nuclei during animal cloning requires extensive DNA demethylation of pluripotency genes such as nanog or oct4, which are silenced in somatic cells by DNA methylation. However, little was known about the molecular mechanisms underlying DNA demethylation. We recently found that Gadd45a is a key regulator of active DNA demethylation of oct4 in Xenopus oocytes and that it acts by promoting DNA repair. Here we propose to study the biological role of Gadd45-mediated DNA demethylation in murine embryonic stem (ES) cells and mouse embryos. While individual Gadd45a, -b and -g knock out mice have been generated previously, these mice are all viable and fertile, likely due to functional redundancy. To circumvent redundancy of Gadd45 genes, we have developed a dominant negative (dn) Gadd45. Using this tool, we will study the role of Gadd45 in pluripotency by overexpressing dnGadd45 (loss of function) as well as wild type Gadd45 (gain of function) in ES cells. In parallel, we will generate and characterize transgenic mice using dnGadd45 as well as wild type Gadd45 and revisit the role of this gene in early mouse embryogenesis.