Nuclear Cloning and the Reprogramming of the Genome

描述（由申请人提供）：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死亡了。这就提出了关于核能力和分化的问题，这些问题在半个世纪前通过将体细胞细胞核移植到卵子中产生第一代两栖动物时就已经提出了。这些问题包括细胞核指导动物形成的效力是否会随着供体细胞年龄的增加而丧失，以及细胞核的分化状态是否会影响克隆中所见的异常类型。核克隆的主要问题被认为是由供体核的错误表观遗传重编程引起的。本研究旨在了解核克隆动物低存活率和异常表型特征的分子机制。我们提出以下项目：我们将通过核移植测试来自供体胚胎和体细胞干细胞的核的效力。2. 我们将生成cDNA阵列，用于建立核重编程的分子标准。3. 我们将设计策略来改善体细胞供体的表观遗传重编程。我们的目标是将体细胞的表观遗传状态转化为类似于胚胎干细胞的状态。

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.