Licensing Factor Expression in Stem Cells

干细胞有助于修复和维持它们所在的组织。这些细胞早已在组织中被识别出来，如血液和上皮细胞，它们快速地翻转。最近，这种细胞的存在也被证明存在于包括大脑在内的被认为没有自我更新能力的器官中。体细胞干细胞与胚胎干细胞一样，在许多应用中被认为是一种潜在的供体细胞来源。此外，这些细胞在组织维持中的作用表明，了解它们在整个生物体中的生物学特性，将广泛应用于理解与年龄相关的功能障碍。然而，一个主要的障碍是，对于大多数组织来说，没有有效的方法来识别或恢复这些细胞。在这里，我们建议利用所有干细胞群共享的一个特性，即它们的细胞分裂潜力，将这些细胞标记为识别、恢复和使用小鼠作为模式生物的遗传操作。这些方法依赖于最近的两项进展。首先，利用绿色荧光蛋白来标记活细胞是一种有效的方法。其次，对细胞周期控制的理解的进步已经确定了更好的基因标记，以保持细胞的增殖能力。这些基因是允许细胞进行DNA复制机制的一部分。这些基因作为干细胞标记手段的优势在于，与以往依赖于s期特异性事件的增殖标记不同，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识别。目前的提案有两个具体的短期目标，旨在解决这一办法在概念上和技术上的可行性。首先，我们将利用脑室下区存在的神经干细胞群的特性来明确确定该组织中大多数或全部缓慢循环的干细胞群是否表达许可因子，从而解决概念上的可行性。其次，我们将利用基因靶向来解决该方法的技术可行性，通过确定从内源性编码Mcm2或其他许可因子表达EGFP荧光时是否易于检测。

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.