Chromatin remodeling and nuclear reprogramming in domestic animal clones and embryonic stem cells

利用克隆技术生产有活力的胚胎、胚胎干细胞系和活体动物已经证实，可以实现分化的胎儿和成体基因组的充分逆转。 然而，胚胎、胎儿和新生儿死亡率高，异常影响胎盘、呼吸和循环系统以及胎儿和新生儿克隆的生长，大大降低了活产率至约1-5%，表明这种重编程过程在大多数情况下是不充分的。 该提案概述了一项持续的研究计划，该计划基于以下假设：人工和次优培养环境改变了细胞和胚胎的染色质结构和功能-这种改变在体外操作和胚胎、胚胎干细胞和克隆程序产生的活产动物的发育过程中被放大并进一步改变。 该研究计划的具体目标是：1）增强供体基因组的核重编程; 2）评估表观遗传和核重编程的效率; 3）比较和对比胚胎克隆和胎儿与其体外受精（IVF）和体内产生的对应物之间的全局基因表达模式。 我们独特的显微操作技能，各种定量基因表达和染色质结构分析工具，以及动物克隆和胚胎干细胞衍生和分化技术在多个国内物种的组合，使我们能够评估体细胞核移植技术的有效性和安全性，并允许改进，以帮助转移这项技术应用的努力。 这项研究计划将提高我们对核重编程事件的基本理解，并可能允许提高家畜克隆的活产率的策略，并对胚胎干细胞在移植治疗中的潜在用途进行遗传评估。

The production of viable embryos, embryonic stem cell lines, and live animals using cloning technologies has confirmed that sufficient reversal of differentiated fetal and adult genomes can be achieved.  However, high embryonic, fetal and neonatal deaths with abnormalities affecting the placenta, the respiratory and circulatory systems and growth of the fetus and neonate clone greatly lower the live birth rate to ~1-5% indicating that this reprogramming process is inadequate most of the time.     This proposal outlines a continuing research program based on the hypothesis that the artificial and sub-optimal culture environments alter the chromatin structure and function of cells and embryos - modifications that are amplified and further altered during the in vitro manipulations and development of embryos, embryonic stem cells and live born animals derived from the cloning procedures.  The specific aims of this research program are to: 1) Enhance nuclear reprogramming of the donor genome; 2) Evaluate the efficiency of epigenetic and nuclear reprogramming; 3) Compare and contrast global gene expression patterns between embryo clones and fetuses with their in vitro fertilized (IVF) and in vivo produced counterparts.     Our unique combination of micromanipulation skills, various quantitative gene expression and chromatin structure analysis tools, and techniques for animal cloning and embryonic stem cell derivation and differentiation in multiple domestic species allow us to evaluate the efficacy and safety of somatic cell nuclear transfer technologies and permits improvements to help transfer this technology to applied endeavours.  This research program will improve our fundamental understanding of nuclear reprogramming events and may allow for strategies that will improve the live birth rates of domestic animal clones and to genetically evaluate embryonic stem stems for their potential uses in transplantation-based therapies.