Nuclear-cytoplasmic interactions in mammalian embryos

基本原理：家畜的繁殖成功率受到妊娠期胚胎死亡率和产后发病率升高的显著影响，特别是在牛和马中，在使用辅助生殖技术如体细胞核移植（SCNT）获得的胚胎和后代中观察到不同水平的基因组印迹异常。此外，虽然细胞重编程已通过表达重编程因子来产生诱导多能干细胞（iPS），但对印迹基因位点的遗传和表观遗传突变的潜在负担知之甚少。因此，需要进一步的研究来阐明和比较马和牛的体细胞重编程的生物学机制。 假设：在受精卵和早期胚胎中普遍存在的全能状态是通过稳定的基因组印记建立的，因此，通过SCNT或iPS细胞程序对体细胞进行真正的重编程只能通过对印记基因座的最小遗传和表观遗传干扰来实现。 目的：阐明和比较不同哺乳动物早期胚胎发育过程中控制印记基因编程的表观遗传和遗传机制，特别是确定使用马和牛杂交模型实现真实体细胞重编程的方法。 具体目标1-探索参与SCNT重编程的细胞和胚胎中印迹基因座重塑的表观遗传机制。 具体目标2-检查在遗传重编程的iPS细胞系及其向体细胞和生殖细胞分化的衍生物中涉及印迹基因座重塑的表观遗传机制。 具体目标3-使用全基因组分析和适当的生物信息学工具研究胚胎和SCNT和iPS细胞重编程衍生的分化细胞中的基因组、甲基化组和转录组变体。 重要性：这些研究将进一步加深我们对哺乳动物早期发育过程中发生的细胞核-细胞质相互作用的理解，并使新的和改进的工具能够确保安全有效地使用细胞重编程方案用于家养物种的生殖和临床应用。

Rationale: Reproductive success in domestic animals is significantly compromised by elevated embryonic mortality during gestation and postnatal morbidity, particularly in bovine and equine where different levels of genomic imprinting abnormalities have been observed in embryos and offspring obtained using assisted reproductive techniques such as somatic cell nuclear transfer (SCNT). Moreover, although cell reprogramming has been achieved by the expression of reprogramming factors to produce induced pluripotent stem (iPS) cells, little is known about the potential burden of genetic and epigenetic mutations to imprinted gene loci. Therefore, further research is needed to elucidate and compare the equine and bovine biological mechanisms involved in the reprogramming of somatic cells. Hypothesis: The totipotent state that prevails in fertilized zygotes and early embryos is established with stable genomic imprints and, therefore, genuine reprogramming of somatic cells either by SCNT or iPS cell procedures can only be achieved through minimal genetic and epigenetic disturbances to imprinted loci. Objective: Elucidate and compare the epigenetic and genetic mechanisms controlling the programming of imprinted genes during early embryonic development in different mammals and particularly identify means of achieving authentic somatic cell reprogramming using equid and bovid hybrid models. Specific objective 1- To explore the epigenetic mechanisms involved in the remodeling of imprinted loci in cells and embryos reprogrammed by SCNT. Specific objective 2- To examine the epigenetic mechanisms involved in the remodeling of imprinted loci in genetically reprogramed iPS cell lines and their differentiated derivatives towards somatic and germ cells. Specific objective 3- To use a whole genome analysis and appropriate bioinformatic tools to investigate genomic, methylomic and transcriptomic variants in embryos and differentiated cell derived by SCNT and iPS cell reprogramming. Significance: These studies will further our understanding of the nuclear-cytoplasmic interactions that take place during early development in mammals and enable novel and improved tools to assure a safe and efficient use of cell reprogramming protocols for reproductive and clinical applications in domestic species.