Genetic and Molecular Approach to Identify Ooplasm Reprogramming Factors

描述（由申请人提供）：本提案是根据RFA， RFA- od -09-003， NIH健康与科学研究挑战基金提交的。广泛的挑战领域是“干细胞”，具体的挑战是14-HD-102：确定卵母细胞的重编程因子。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依赖于细胞核重编程的能力。卵母细胞是唯一能够将体细胞细胞核重新编程为胚胎，全能性状态的细胞，尽管在支持足月发育方面成功率很低。这种能力可能被用来制造干细胞。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编程的特定卵母细胞因子，从而可以操纵卵母细胞的重编程能力来改善克隆，并且可以利用这些相同的因子来推进干细胞技术。然而，卵母细胞中可能有数百种影响染色质结构和基因转录的因素，但其中只有少数可能与重编程有关。因此，简单地对卵母细胞中表达的潜在重编程因子进行分类价值有限。需要明确表达的基因与细胞重编程能力的关系。遗传系统可以将性状的变异与基因定位数据和阵列表达差异的组合联系起来，为规避这些限制提供了无与伦比的机会。基因定位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。我们有一种基因系统，非常适合这个目的。我们已经证明，用C57BL/6 （B6）卵子制造的克隆比用D2卵子制造的克隆更有效地超越了2细胞阶段；F1杂交卵支持较高的发育速率，表明杂交活力效应。引导克隆胚胎发育超过2细胞阶段的能力是卵母细胞重编程潜力的明确指标。因此，与B6和F1杂交体相比，D2卵母细胞在重编程体细胞细胞核以支持早期胚胎发生方面较差。我们将使用B6xD2重组自交系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因子的阵列表达数据相结合，以确定候选基因，然后进行功能研究，以确定哪些基因决定了卵母细胞的重编程能力。这种结合遗传和分子的方法，建立在表型差异的基础上，因此将导致鉴定新的重编程因子。

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.