Germline-specific reprogramming in cloned mice

描述(申请人提供)：在雄性和雌性哺乳动物中，配子发生都是一个复杂的过程，包括遗传和表观遗传编程机制，这些机制使配子基因组准备好指导后续胚胎的发育。当克隆的个体通过将体细胞核转移到去核的卵子中而产生时，供体基因组没有经历配子发生所特有的任何重编程过程。因此，移植的体细胞核必须对来自受体卵质的信号做出反应，指示它经历快速的重新编程，以促进胚胎发育。在最近的研究中，对克隆小鼠体细胞表观遗传重编程的程度进行了部分分析，发现这些重编程是不完整的和高度可变的。然而，还没有研究确定在克隆小鼠的生殖细胞中恢复表观遗传编程的程度。此外，以前还没有关于克隆小鼠中任何细胞类型的遗传编程的研究。在这一应用中，我们提出了一项系统的研究，以比较通过核移植(NT)方法克隆产生的胚胎和后代的体细胞和生殖细胞中遗传参数和表观遗传参数变得适当重新编程的程度。只有一小部分克隆胚胎(不到2-3%)发育到足月，而且只有一小部分克隆胚胎成长为可生育的成年胚胎。我们假设，虽然遗传和表观遗传编程在克隆小鼠的体细胞中是可变的和不完整的，但在这些个体的种系中可以在很大程度上恢复。我们进一步怀疑，为了克隆胚胎的正常发育，供体核必须经历低频率突变的表观遗传机制的快速预编程。最后，我们怀疑来自体细胞和生殖细胞的核在它们的遗传和表观遗传编程的初始状态上是不同的，因此它们在NT之后成功地指导胚胎发育的能力不同。这些实验的结果将有助于两个普遍感兴趣的领域。首先，它们将使我们更好地了解遗传和表观遗传重编程的种系特有机制如何正常发挥作用。其次，它们将为哺乳动物克隆的有效性和安全性提供重要的新见解。

DESCRIPTION (provided by applicant): In both male and female mammals, gametogenesis is a complex process that includes genetic and epigenetic programming mechanisms that prepare the gametic genomes to direct development of an ensuing embryo. When a cloned individual is produced by transfer of a somatic cell nucleus into an enucleated egg, the donor genome has NOT undergone any of the reprogramming processes unique to gametogenesis. Thus the transplanted somatic cell nucleus must respond to signals from the recipient ooplasm directing it to undergo rapid reprogramming to facilitate embryonic development. In recent studies the extent of epigenetic reprogramming in somatic cells of cloned mice has been partially analyzed, and found to be incomplete and highly variable. However, no studies have been carried out to determine the extent to which epigenetic programming is restored in germ cells of cloned mice. Furthermore, there have been no previous studies of genetic programming in any cell type in cloned mice. In this application, we propose a systematic study to compare the extent to which genetic and epigenetic parameters become properly reprogrammed in somatic and germ cells of embryos and offspring produced by cloning via the nuclear transfer (NT) method. Only a small proportion (less than 2-3%) of cloned embryos develop to term, and only a subset of these grow into fertile adults. We hypothesize that while genetic and epigenetic programming is variable and incomplete in somatic cells of cloned mice, it can be largely restored in the germline of these individuals. We further suspect that for a cloned embryo to develop normally, the donor nucleus must undergo rapid preprogramming of epigenetic mechanisms with a low frequency of mutations. Finally, we suspect that nuclei from somatic cells and germ cells differ in their initial states of genetic and epigenetic programming and hence in their ability to successfully direct embryonic development following NT. The results of these experiments will contribute to two general areas of interest. First, they will allow us to better understand how germline-specific mechanisms of genetic and epigenetic reprogramming normally function. Second, they will provide important new insight into the efficacy and safety of mammalian cloning.