Genetic and Molecular Approach to Identify Ooplasm Reprogramming Factors

描述(由申请人提供)：本提案是为响应RFA、RFA-OD-09-003、NIH健康与科学研究挑战基金而提交的。广泛的挑战领域是“干细胞”，而具体的挑战是14-HD-102：识别卵母细胞的重编程因素。干细胞治疗疾病和修复组织损伤的新方法的出现是最近科学中最令人兴奋的发展之一。一些最令人兴奋的干细胞技术取决于对细胞核进行重新编程的能力。卵母细胞独一无二地能够将体细胞核重新编程为胚胎的全能状态，尽管在支持长期发育方面成功率很低。这种力量可能被利用来衍生干细胞。追求这些令人兴奋的可能性的一个关键目标是发现驱动核重新编程的特定卵母细胞因素，以便可以操纵卵母细胞的重新编程能力来改进克隆，从而利用这些相同的因素来推进干细胞技术。然而，卵母细胞中可能有数百个因素影响染色质结构和基因转录，但只有少数因素可能与重新编程有关。因此，简单地对在卵母细胞中表达的潜在的重新编程因子进行分类是有限的。表达的基因与细胞的重新编程能力之间需要明确的关系。基因系统可以将一个性状的变异与基因图谱数据和阵列表达差异的组合相关联，这为规避这些限制提供了无与伦比的机会。基因作图数据可以极大地促进阵列数据的解释，并且将阵列数据与表型的不同变体相关联同样具有很高的信息量。我们已经有了一种非常适合这一目的的基因系统。我们已经证明，用C57BL/6(B6)卵制成的克隆比用D2卵制成的克隆在2-细胞期后进展得更有效；F1杂交卵支持更高的静止发育率，这表明了杂交活力效应。指导克隆胚胎发育超过2-细胞期的能力是卵母细胞重新编程潜力的一个明显指标。因此，与B6和F1相比，D2卵母细胞在重新编程体细胞核以支持早期胚胎发生方面表现较差。我们将使用B6xD2重组近交系菌株来确定导致这种差异的重编程因子基因的数量和染色体位置。我们将把这些数据与已知转录因子和染色质调节剂的阵列表达数据结合起来，以确定候选基因，然后进行功能研究，以确认哪些基因决定卵母细胞的重编程能力。因此，这种建立在表型差异基础上的遗传和分子相结合的方法将导致识别新的重新编程因素。 与公共卫生相关：在克隆过程中确定卵子中负责核编程的因素非常有兴趣，因为这种知识可能导致改进产生干细胞的方法，并为一系列基本和应用目的克隆动物。困难在于如何确定鸡蛋中无数表达的转录因子和染色质调节因子中的哪一种负责小鼠的重编程能力，我们已经获得了相关小鼠品系的丰富的阵列表达数据。我们将在这里结合这些数据来确定基因定位、基因表达和功能研究，这些基因决定了卵母细胞重编程能力，从而成为关键的卵母细胞重编程因素。

DESCRIPTION (provided by applicant): This proposal is submitted in response to the RFA, RFA-OD-09-003, NIH Challenge Grants in Health and Science Research. The Broad Challenge Area is "Stem Cells", and the specific Challenge is 14-HD-102: Identifying Reprogramming Factors for Oocytes. The advent of new stem cell approaches to cure disease and repair tissue damage is one of the most exciting developments in recent science. Some of the most exciting stem cell technology rests with the ability to reprogram nuclei. The oocyte is uniquely able to reprogram somatic cell nuclei to an embryonic, totipotent state, albeit with a low percentage of success in supporting term development. This power may be harnessed to derive stem cells. A key goal in pursuit of these exciting possibilities is to discover the specific oocyte factors that drive nuclear reprogramming, so that the reprogramming capacity of the oocyte can be manipulated to improve cloning, and so that those same factors can be exploited to advance stem cell technologies. However, there may be hundreds of factors in the oocyte that affect chromatin structure and gene transcription, but only a few of these may be relevant to reprogramming. Thus, simply cataloguing potential reprogramming factors that are expressed in an oocyte is of limited value. A clear relationship of expressed genes to reprogramming capacity of a cell is needed. Genetic systems that can correlate variations in a trait with a combination of gene mapping data and array expression differences offer unparalleled opportunity for circumventing such restrictions. The gene mapping data can greatly facilitate the interpretation of array data, and correlating array data with different variants in phenotype is likewise highly informative. We have available a genetic system that is ideal for this purpose. We have shown that clones made with C57BL/6 (B6) eggs progress beyond the 2-cell stage much more efficiently than those made with D2 eggs; F1 hybrid eggs support a higher still rate of development indicative of a hybrid vigor effect. Ability to direct cloned embryo development beyond the 2-cell stage is a clear indicator of oocyte reprogramming potential. Thus, D2 oocytes are inferior at reprogramming somatic cell nuclei to support early embryogenesis compared to B6 and F1 hybrids. We will employ B6xD2 recombinant inbred strains to determine the number and chromosomal locations of reprogramming factor genes that account for this difference. We will combine those data with array expression data for known transcription factors and chromatin regulators to identify candidates, and then perform functional studies to confirm which genes determine reprogramming capacity of the oocyte. This combined genetic and molecular approach, built on a foundation of phenotype difference will thus result in identification of novel reprogramming factors. PUBLIC HEALTH RELEVANCE: There is great interest in identifying factors in the egg that are responsible for nuclear programming during cloning, because such knowledge may lead to enhanced methods for generating stem cells, and for cloning animals for a range of basic and applied purposes. The difficulty is how to determine which of the myriad of expressed transcription factors and chromatin regulators in the egg are responsible for reprogramming capacity in mice, and we have obtained already a wealth of array expression data for the relevant mouse strains. We will combine these data here to identify gene mapping, gene expression, and functional studies those specific genes that determine oocyte reprogramming capacity, and hence serve as key oocyte reprogramming factors.