Male germline stem cell culture and genetic modification

描述(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(SSCs)生物学以及精子发生过程中分化过程的系统。目前还没有生化、分子或形态标准来鉴定SSC，只有功能分析才能确定SSC在任何细胞群体中的存在。在过去的八年中，移植技术已经证明，干细胞可以被超低温保存，可以从许多物种移植，在体外保持，并经过基因修改。这项技术还被用于建立负责自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计时由生殖细胞的遗传程序控制。因此，移植系统为研究干细胞、其在生精小管中的位置以及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，但从成熟动物的睾丸中获取和研究SSCs的纯群体是不可能的，这极大地阻碍了对这些细胞生物学的研究。我们建议在小鼠身上进行一系列实验，以进一步丰富睾丸细胞群的干细胞含量，然后使用这些纯化的群将遗传修饰引入SSCs。我们计划检查的一组重要基因是那些已被证明能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体：1)直接从动物身上分离的睾丸细胞群体，极大地丰富了干细胞；2)永生化细胞系，可以在体外作为干细胞或早期精原细胞进行繁殖。这些细胞在受体动物的生精小管上定植并产生精子的能力以及它们在体外分化的能力将被用来表征原始细胞群并研究分化过程。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了解SSCs生物学和精子发生的目标前进，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.