Developmental Reprogramming after Nuclear Transfer

描述（由申请人提供）：受精卵发育为复杂生物体传统上被认为是一个单向过程，发育中的胎儿细胞逐渐趋向于特定的组织类型。哺乳动物核移植克隆的最新进展表明，哺乳动物卵母细胞具有显著的能力，可以解除细胞分化的限制，使成体细胞核恢复到全能的胚胎状态。因此，NT克隆提供了一个独特的机会来阐明成熟细胞可以恢复到未分化状态的分子和细胞机制，这一过程称为发育重编程。 目的1）确定能否从终末分化细胞直接克隆小鼠。克隆小鼠仅使用胚胎干（ES）细胞中间体从终末分化细胞产生，这表明细胞核通过ES细胞可能是完全重编程所必需的。将进行实验以确定这是否是真的，或者卵母细胞和胚胎单独是否可以成功地重新编程终末分化细胞核的表观遗传状态。 目的2）研究NT后基因组转录活性的变化是否可以解释从分化状态到多能状态的转变。为此，基因组范围内的变化，转录活性发生后NT正在评估微阵列分析。将进行实验来表征这些观察到的受精和克隆的植入前胚胎之间的基因表达变化的功能重要性。这里提出的实验结合了联合收割机的分子，遗传和发育的方法，将测试的限制重编程，阐明机制管理重编程和确定如何在重编程的不足可能导致克隆的效率低下的性质。这些研究可能对评估NT技术的医疗效用、破译多能性的分子基础以及扩大我们对胚胎发育和干细胞生物学的理解具有深远的重要意义。

DESCRIPTION (provided by applicant): The development of the fertilized zygote into a complex organism has traditionally thought to be a unidirectional process, with cells in the developing fetus becoming gradually more committed to a specific tissue type. The recent development of mammalian cloning by nuclear transfer (NT) suggests that the mammalian oocyte has the remarkable ability to relieve the constraints imposed by cellular differentiation and return an adult nucleus to a totipotent, embryonic state. Thus, cloning by NT provides a unique opportunity to elucidate the molecular and cellular mechanisms by which an adult cell can be returned to an undifferentiated state, a process termed developmental reprogramming. Aim 1) To determine whether mice can be cloned directly from terminally differentiated cells. Cloned mice have only been generated from terminally differentiated cells using an embryonic stem (ES) cell intermediate, suggesting that passage of the nucleus through an ES cell might be necessary for complete reprogramming. Experiments will be carried out to determine whether this is true or if instead, the oocyte and embryo alone can successfully reprogram the epigenetic state of a terminally differentiated nucleus. Aim 2) To investigate if the transition from a differentiated state to a pluripotent state can be understood through the genome-wide changes in transcriptional activity taking place after NT. Towards this end, the genome wide changes in transcriptional activity taking place after NT are being assessed by microarray analysis. Experiments to characterize the functional importance of these observed gene expression changes between fertilized and cloned preimplantation embryos will be carried out. The experiments proposed here combine molecular, genetic and developmental approaches that will test the limits of reprogramming, elucidate mechanisms governing reprogramming and determine how inadequacies in reprogramming may lead to the inefficient nature of cloning. These studies may have profound importance for evaluating the medical utility of NT technology, deciphering the molecular basis of pluripotency and expanding our understanding of embryonic development and stem cell biology.