Male germline stem cell culture and genetic modification

描述（由申请人提供）：睾丸细胞移植技术的发展建立了一个研究精原干细胞（SSC）生物学以及精子发生过程中发生的分化过程的系统。目前，没有生物化学、分子或形态学标准可以鉴定SSC，只有功能测定可以确定SSC在任何细胞群中的存在。在过去的八年中，移植技术已经证明，干细胞可以冷冻保存，从许多物种移植，在体外维持，并进行遗传修饰。该技术也已被用于建立负责天然和诱导基因突变的细胞类型（生殖细胞或支持细胞），并证明精子发生期间的细胞周期时间由生殖细胞的遗传程序控制。因此，移植系统为研究干细胞及其在生精小管中的生态位和精子发生过程提供了一种强有力的方法。 尽管取得了这些进展，但不可能从成熟动物的睾丸中获得和研究纯的精原干细胞群，这极大地阻碍了对这些细胞生物学的研究。我们建议在小鼠中进行一系列实验，以进一步富集睾丸细胞群的干细胞含量，然后使用这些纯化的群体将遗传修饰引入SSC中。我们计划研究的一组重要的基因是那些已经证明可以使干细胞永生的基因。这种方法将为我们提供两种生殖细胞群：1）直接从动物中分离的睾丸细胞群，并大大富集干细胞，2）可以作为干细胞或早期精原细胞在体外增殖的永生化细胞系。这些细胞定殖受体动物的生精小管和产生精子发生的能力以及它们在体外分化的能力将用于表征原始细胞群和研究分化过程。在过去的八年里，移植技术在我们的研究中一直是一种富有成效的方法，并已被证明对其他科学家越来越有价值。概述的研究将继续朝着我们理解精原干细胞和精子发生的生物学的目标取得进展，我们的许多发现将适用于其他物种。

DESCRIPTION (provided by applicant): Development of the testis cell transplantation technique established a system to study the biology of spermatogonial stem cells (SSCs) as well as the differentiation processes that occur during spermatogenesis. Currently there are no biochemical, molecular or morphological criteria by which the SSC can be identified, and only a functional assay can establish the presence of the SSC in any cell population. During the past eight years the transplantation technique has demonstrated that the stem cell can be cryopreserved, transplanted from many species, maintained in vitro, and subjected to genetic modification. The technique also has been used to establish the cell type (germ cell or Sertoli cell) responsible for both natural and induced genetic mutations, and to demonstrate that cell cycle timing during spermatogenesis is controlled by the genetic program of the germ cell. Thus, the transplantation system has provided a powerful approach to study the stem cell, its niche in the seminiferous tubule, and the process of spermatogenesis. Despite these advances, it is not possible to obtain and study pure populations of SSCs from testes of mature animals, which greatly impedes studies on the biology of these cells. We propose a series of experiments in the mouse to further enrich testis cell populations for stem cell content and then to use these purified populations for introduction of genetic modifications into the SSCs. An important group of genes we plan to examine for their effect are those that have been shown to immortalize stem cells. This approach will provide us with two populations of germ cells: 1) testis cell populations isolated directly from the animal and greatly enriched for stem cells, and 2) immortalized cell lines that can be propagated in vitro as stem cells or early stage spermatogonia. The ability of these cells to colonize the seminiferous tubules of recipient animals and generate spermatogenesis as well as their ability to differentiate in vitro will be used to characterize the original cell populations and to study the differentiation process. The transplantation technique has been a productive approach in our studies over the past eight years and has proven increasingly valuable to other scientists. The studies outlined will continue progress toward our objective of understanding the biology of SSCs and spermatogenesis, and many of our findings will be applicable to other species.