Male germline stem cell culture and genetic modification

雄性生殖干细胞培养和基因改造

• 批准号: 7228882
• 负责人: Ralph Lawrence Brinster
• 金额: $ 33.81万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7228882
• 关键词: Actins; Adult; Affect; Animals; Biochemical; Biological Assay; Biology; Cell Cycle; Cell Line; Cell Separation; Cell Transplantation; Cells; Cellular biology; Chickens; Dependovirus; Development; Doxycycline; Elongation Factor; Fluorescence-Activated Cell Sorting; Galactosidase; Gene Mutation; Genes; Genetic; Genetic Programming; Germ Cells; Glycoproteins; Green Fluorescent Proteins; In Vitro; LacZ Genes; Magnetism; Methods; Modification; Molecular; Moloney Leukemia Virus; Mus; Phosphoglycerate Kinase; Population; Process; Reporter; Scientist; Seminiferous tubule structure; Series; Spermatogenesis; Spermatogonia; Staging; Stem cells; System; Techniques; Testis; Tetanus Helper Peptide; Tetracycline; Tetracyclines; Time; Transplantation; Vesicular stomatitis Indiana virus; Virus; analog; cell type; immortalized cell; male; mature animal; promoter; pup; research study; sertoli cell

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.

描述（由申请人提供）：睾丸细胞移植技术的开发建立了一种系统，以研究精子干细胞（SSC）的生物学以及精子发生过程中发生的分化过程。目前尚无生化，分子或形态学标准，可以通过这些标准来识别SSC，并且只有功能分析才能在任何细胞群中建立SSC的存在。在过去的八年中，移植技术表明，可以冷冻保存干细胞，从许多物种移植，维持体外并进行遗传修饰。该技术还被用于建立负责自然和诱导遗传突变的细胞类型（生殖细胞或Sertoli细胞），并证明生殖细胞的遗传学程序控制了精子发生过程中细胞周期的时机。因此，移植系统提供了一种强大的方法来研究干细胞，其生殖小管中的细胞和精子发生过程。 尽管取得了这些进步，但不可能从成熟动物的睾丸中获得和研究SSC的纯种群，这极大地阻碍了对这些细胞的生物学的研究。我们在小鼠中提出了一系列实验，以进一步富集干细胞含量的睾丸细胞群，然后使用这些纯化的群体将遗传修饰引入SSC中。我们计划检查其作用的一组重要基因是已证明使干细胞永生的基因。这种方法将为我们提供两个生殖细胞的种群：1）直接从动物中分离出来的睾丸细胞群体，并极大地富集了干细胞，以及2）可以在体外作为干细胞或早期精子的永生细胞系。这些细胞在受体动物和生成精子发生及其在体外分化的能力中定居的能力将用于表征原始细胞群体并研究分化过程。在过去的八年中，移植技术在我们的研究中一直是一种富有成效的方法，事实证明，对其他科学家来说越来越有价值。概述的研究将继续朝着我们了解SSC的生物学和精子发生的目标，我们的许多发现将适用于其他物种。