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为研究精原干细胞（ssc）的生物学以及精子发生过程中的分化过程建立了一个系统。目前还没有生物化学、分子或形态学标准来鉴定SSC，只有功能测定才能确定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，并进行基因改造。该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），并证明精子发生过程中的细胞周期时间是由生殖细胞的遗传程序控制的。因此，移植系统为研究干细胞及其在精管中的生态位和精子发生过程提供了强有力的途径。

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.