Nuclear Cloning and the Reprogramming of the Genome

描述(申请人提供)：在所有克隆成功的哺乳动物物种中，最多只有百分之几的核移植胚胎发育到足月，其中许多在出生后不久就会死亡。这引发了半个世纪前通过将体细胞核移植到卵子中产生第一代两栖动物时提出的核潜力和分化问题。这些问题包括细胞核指导动物形成的效力是否随着供体细胞年龄的增加而丧失，以及其分化状态是否会影响克隆中看到的异常类型。核克隆的主要问题被认为是供体核的表观遗传重新编程错误造成的。这一建议试图了解导致核克隆动物存活率低和表型特征异常的分子机制。我们提出了以下项目：1.通过核移植检测供体胚胎和体细胞来源的核的效力。2.我们将生成用于建立核重新编程的分子标准的cdna阵列。3.我们将设计策略来改善体细胞供体细胞的表观遗传重编程。我们的目标是将体细胞的表观遗传状态转换为类似于ES细胞的状态。 4.我们将从成熟的神经元供体细胞克隆小鼠。这将测试神经元基因组是否会发生变化，作为正常大脑生理的一部分，这将限制核能力。胚胎和成人干细胞被认为为再生细胞治疗提供了巨大的潜力。然而，应用这一有希望的方法的主要问题尚未解决。例如，胚胎和成人干细胞核的表观遗传状态可能比终末分化细胞的表观遗传状态更容易重新编程。这项提议的中心焦点是建立区分核与干细胞和分化细胞的表观遗传状态的功能和生物学参数。这将作为改变体细胞潜能的分子基础，以便它们可以被重新编程为不同类型的细胞，可以用于治疗方法。事实上，如果能够理解导致重新编程的分子机制，最终可能会操纵体细胞并产生类似ES细胞的细胞，而不需要卵母细胞和核移植。

DESCRIPTION (provided by applicant): In all mammalian species where cloning has been successful, at best a few percent of nuclear transfer embryos develop to term, and of those many die shortly after birth. This has raised questions of nuclear potency and differentiation that were posed half a century ago with the generation of the first amphibians by transfer of a somatic nucleus into the egg. These issues include whether the potency of a nucleus to direct the formation of an animal is lost with increasing age of the donor cell and whether its state of differentiation influences the type of abnormalities seen in the clone. The main problem of nuclear cloning is thought to be caused by faulty epigenetic reprogramming of the donor nucleus. This proposal seeks to understand molecular mechanisms that are responsible for the low survival rate and abnormal phenotypic characteristics of animals derived by nuclear cloning. We propose the following projects: 1. We will test the potency of nuclei derived from donor embryonic and somatic stem cells by nuclear transfer. 2. We will generate cDNA arrays that will be used to establish molecular criteria for nuclear reprogramming. 3. We will design strategies to improve the epigenetic reprogramming of somatic donor cells. Our goal is to convert the epigenetic state of the somatic cell to one that resembles that of an ES cell. 4. We will clone mice from mature neuronal donor cells. This will test whether alterations occur in the genome of neurons as part of normal brain physiology that would restrict nuclear potency. Embryonic and adult stem cells are thought to offer significant potential for regenerative cell therapy. However, major issues of applying this promising approach are unresolved. For example, the epigenetic state of embryonic and adult stem cell nuclei may be more amenable to reprogramming than that of terminally differentiated cells. It is a central focus of this proposal to establish functional and biological parameters that distinguish the epigenetic state of nuclei from stem cells and differentiated cells. This will serve as a molecular basis for altering the potential of somatic cells so they could be reprogrammed into different cell types that can be used in therapeutic approaches. Indeed, if the molecular mechanisms that are responsible for reprogramming could be understood, it might eventually be possible to manipulate a somatic cell and generate an ES cell-like cell without the need for oocytes and nuclear transfer.