Licensing Factor Expression in Stem Cells

干细胞有助于修复和维护它们所处的组织。 这些细胞长期以来一直在组织中被识别，如血液和上皮细胞，它们迅速翻转。 最近，这种细胞的存在也被证明存在于被认为不能自我更新的器官中，包括大脑。 体细胞来源的干细胞与胚胎干细胞一样，在许多应用中被认为是潜在的供体细胞来源。 此外，这些细胞在组织维护中的作用表明，了解它们在整个生物体中的生物学特性将对了解年龄相关功能障碍具有广泛的应用。 然而，一个主要的障碍是，对于大多数组织，没有有效的方法来识别或回收这些细胞。 在这里，建议利用所有干细胞群体共有的一个特性，即它们的细胞分裂潜力，以小鼠为模型生物标记这些细胞进行鉴定、恢复和遗传操作。 这些方法依赖于两项最新进展。 首先，现在可以使用绿色荧光蛋白来标记活细胞。 其次，在细胞周期控制的理解的进步已经确定了更好的基因标记的细胞保留的增殖能力。 这些基因是允许细胞进行DNA复制的机制的一部分。 这些基因作为标记干细胞的方法的优点在于，与依赖于S期特异性事件的先前增殖标记物不同，这些因子在G1期或整个细胞周期中表达。 此外，初步研究表明它们在大多数分化细胞中不表达。 因此，任何能够增殖的细胞，而不仅仅是那些正在进行DNA合成的细胞，都可以通过这些基因的表达来鉴定。本提案有两个具体的短期目标，旨在解决这一方法的概念和技术可行性。 首先，我们将解决概念上的可行性，利用大脑室管膜下区的神经干细胞群的属性，以明确确定是否大多数或所有的缓慢循环的干细胞群，该组织表达许可因子。 第二，我们将利用基因靶向，以解决技术的可行性的方法，通过确定是否EGFP荧光是容易检测时，从内源性编码Mcm2或替代许可的因素表达。

Stem cells contribute to the repair and maintenance of the tissues in which they reside. These cells have long been recognized in tissues, such as blood and epithelia, which turn over rapidly. More recently, the presence of such cells has also been demonstrated in organs that were not thought to be capable of self-renewal including the brain. Stem cells from somatic source, like their embryonic counter parts, are recognized as a potential source of donor cells in many applications. Additionally, the role of these cells in tissue maintenance suggest that understanding their biology, in the whole organism, will have wide application to understanding age related dysfunction. However, a major obstacle has been that, for most tissues, there is no effective means of identifying or recovering these cells. Here it is proposed to take advantage of one property which all stem cell populations share, their potential for cell division, to mark these cells for identification, recovery, and genetic manipulation using the mouse as a model organism. The methods rely on two recent advances. First, an effective means pf marking living cells, using green fluorescent protein, is now available. Secondly, advances in the understanding of cell-cycle control have identified better gene markers for cells which retain the capacity for proliferation. These genes are part of the mechanism which licenses cells for DNA replication. The advantage of these genes as a means of marking stem cells is that, unlike previous markers for proliferation that relied on S-phase specific events, these factors are expressed in G1 or throughout the cell cycle. Further, preliminary studies demonstrate that they are not expressed in most differentiated cells. Consequently, any cell which is capable of proliferating, not just those undergoing DNA synthesis, can be identified by expression of these genes.. There are two specific short term objectives of the current proposal that aim to address the conceptual and technical feasibility of this approach. First, we will address the conceptual feasibility by taking advantage of the properties of the neural stem cell population present within the subventricular zone of the cerebrum to definitively determine whether most or all of the slowly cycling stem cell population of this tissue expresses licensing factor. Second we will utilize gene targeting to address the technical feasibility of the approach by determining whether EGFP fluorescence is readily detectable when expressed from the endogenous encoding Mcm2 or alternative licensing factors.