Induction of pluripotency by protein gain of function in cloned mouse embryos

与其他重编程方法相比，体细胞核移植到卵母细胞依赖于自然存在的细胞，并且具有快速和全能的特点。由于所得到的多能干细胞质量优越，并且许多卵母细胞的mRNAs在卵母细胞蛋白质组中找不到匹配的，我们提出了假设：1)卵母细胞重编程因子不同于ES和iPS细胞中的已知因子；2)卵母细胞介导的重编程也使用新的蛋白质，这些蛋白质是在卵母细胞激活后立即合成的通用信息。我们预测这些卵母细胞因子将与已知的iPS重编程因子协同作用，导致体细胞基因组更完整的表观遗传‘重置’。因此，我们提出了两组实验。首先，我们将结合生物化学、质谱学和生物信息学来确定激活的小鼠卵母细胞原核中存在的候选重编程因子。目的对中期II期卵母细胞蛋白质组进行筛选，保留了3699种蛋白质中的29种。其次，我们将在小鼠成纤维细胞转化为iPS细胞的过程中表达旧的(29)和新的候选基因。在有无卵母细胞因子的情况下，通过比较Nanog基因诱导率和多能细胞集落形成率来评估重编程效率的预期收益。我们的项目至少在三个方面非常有创新性：首先，它将定义在卵母细胞中运作的自然重编程因子的目录。其次，我们的项目将是第一次通过卵母细胞培养来寻找iPS重新编程的限制阶段的尝试。第三，与重新编程功能无关，我们的蛋白质清单将为成熟女性生殖细胞的分子格局带来新的洞察力。

Compared to the other reprogramming methods, somatic cell nuclear transfer into oocytes relies on a naturally existing cell, and is fast and totipotent. Since the resultant pluripotent stem cells are of superior quality, and many oocytic mRNAs find no match in the oocyte proteome, we put forward the hypotheses that: 1) the oocytic reprogramming factors differ from those known in ES and iPS cells; and 2) oocyte-mediated reprogramming uses also new proteins that are General information synthesized immediately after oocyte activation. We predict these oocytic factors to synergize with the known iPS reprogramming factors, leading to a more complete epigenetic 'resetting' of the somatic genome. Accordingly, we propose two sets of experiments. Firstly, we will combine biochemistry, mass spectrometry and bioinformatics to identify candidate reprogramming factors present in the pronuclei of activated mouse oocytes. Objective screening criteria have already been tested on the proteome of metaphase II oocytes, retaining 29 of 3699 proteins. Secondly, we will express the old (29) and new candidates in mouse fibroblasts during their conversion to iPS cells. The expected gain in reprogramming efficiency will be assessed by comparing rates of Nanog gene induction and pluripotent cell colony formation in the presence vs absence of the oocytic factor. Our project is very innovative on at least three fronts: firstly, it will define the catalog of natural reprogramming factors that operate in the oocyte. Secondly, our project will be the first oocyteeducated attempt to find the limiting stage of iPS reprogramming. Thirdly, independently of reprogramming function, our list of proteins will bring new insight in the molecular landscape of the mature female germ cell.