Genetic and Molecular Approach to Identify Ooplasm Reprogramming Factors

鉴定卵质重编程因子的遗传和分子方法

• 批准号: 7944163
• 负责人: Keith E Latham
• 金额: $ 50万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7944163
• 关键词: Accounting; Affect; Agriculture; Animals; Area; Biological Preservation; Biological Products; Cataloging; Catalogs; Cell Nucleus; Cells; Chromatin; Chromatin Structure; Chromosome Mapping; Chromosomes; Cloning; Companions; Data; Development; Disease; Embryo; Embryonic Development; Experimental Animal Model; Foundations; Gene Expression; Gene-Modified; Genes; Genetic; Genetic Transcription; Goals; Grant; Hand; Hybrid Vigor; Hybrids; Inferior; Knowledge; Lead; Location; Maps; Methods; Molecular Genetics; Mouse Strains; Mus; Nuclear; Oocytes; Ooplasm; Outcome; Patients; Performance; Phenotype; Process; Production; Property; Recombinant Inbred Strain; Recombinants; Rest; Role; Science; Somatic Cell; Staging; Stem cells; Surveys; System; Transcription factor genes; United States National Institutes of Health; Variant; Wound Healing; animal cloning; egg; health science research; improved; interest; novel; nuclear reprogramming; programs; public health relevance; response; stem cell technology; success; trait; transcription factor

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.

描述（由申请人提供）：该提案是根据RFA，RFA-OD-09-003，NIH NIH挑战卫生和科学研究的赠款提交的。广泛的挑战区域是“干细胞”，具体挑战是14-HD-102：识别卵母细胞的重编程因子。新的干细胞方法来治愈疾病和修复组织损伤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重新编程核的能力。卵母细胞具有独特的能力，能够将体细胞核重新编程为胚胎，全能状态，尽管在支持期限发展方面的成功率很低。该功率可以利用以得出干细胞。追求这些令人兴奋的可能性的关键目标是发现驱动核重编程的特定卵母细胞因素，以便可以操纵卵母细胞的重编程能力以改善克隆，以便可以利用这些相同的因素来推进干细胞技术。但是，影响染色质结构和基因转录的卵母细胞中可能有数百个因素，但其中只有少数可能与重编程有关。因此，仅简单地将卵母细胞表达的潜在重编程因子分解为有限的价值。需要明确的基因与细胞重编程能力的明确关系。可以将性状变化与基因映射数据和阵列表达差异的组合相关的遗传系统为规避这种限制提供了无与伦比的机会。基因映射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中的不同变体相关联同样有用。我们有一个遗传体系，非常适合此目的。我们已经表明，用C57BL/6（B6）卵制成的克隆超出了2细胞阶段的效率要比D2鸡蛋制成的克隆更有效。 F1混合卵支持更高的静止发展速率，以表明杂种活力效应。能够将克隆的胚胎发育直接超出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杂种相比，在重编程体细胞核的重编程中，D2卵母细胞支持早期的胚胎发生。我们将采用B6XD2重组近交菌株来确定重新编程因子基因的数量和染色体位置，以解释这种差异。我们将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相结合，以识别候选者，然后执行功能研究以确认哪些基因确定卵母细胞的重编程能力。因此，这种结合的遗传和分子方法建立在表型差异的基础上，将导致鉴定新的重编程因子。 公共卫生相关性：在克隆过程中识别负责核编程的因素非常有兴趣，因为这样的知识可能会导致生成干细胞的方法增强，并出于针对一系列基本和应用的目的而克隆动物。困难是如何确定鸡蛋中的多种表达转录因子和染色质调节剂中的哪些是负责小鼠的重编程能力，并且我们已经获得了相关小鼠菌株的大量阵列表达数据。我们将在此处结合这些数据，以确定那些确定卵母细胞重编程能力的特定基因的基因映射，基因表达和功能研究，因此是关键的卵母细胞重编程因子。