PURIFYING HOMOGENEOUS SPERMATID SUB-POPULATIONS

描述（改编自申请人的描述）：精子发生是一种 顺序组装过程的基础上仔细定时翻译存储 转录沉默细胞中的mRNA;然而， 决定单个mRNA何时被翻译的基因 明白问题的很大一部分是由于我们没有可靠的 纯化不同发育阶段精子细胞亚群的方法 成熟用于分子分析。这项研究旨在解决这一问题。 缺陷 申请人提出开发一种使用转基因技术的方法， 小鼠，表达水母绿色荧光蛋白（GFP）后， 减数分裂雄性生殖细胞，与荧光激活细胞结合 分选（FACS），以纯化分阶段精子细胞的同质亚群。 本研究的目标是：1）分离至少20个精子细胞亚群， 通过流式细胞仪。 2)评估精子细胞的纯度和阶段 基于形态学，超微结构， 和分子标准;选择10个同质亚群， 精子形成的不同阶段3)纯化mRNP颗粒- 相关的mRNA（储存信息）和多核糖体相关的mRNA（活性 信息），制备cDNA 从每个mRNA样品的文库中扩增，并通过RT-PCR评估每个。这项工作将 建立一个可靠的精子细胞分选方案， 大部分或全部活性和储存的精子发生信息的文库。 我们的长期目标是使用这项技术来编目和表征 大多数或所有生精mRNAs，并确定何时使用每一个， 精子成熟 在目前提交的申请中，申请人要求两个 多年的R 03资金，以严格建立技术， 研究，以及无数其他关于精子发生的分子研究， 可以建立。

DESCRIPTION (Adapted from applicant's description): Spermiogenesis is a sequential assembly process based on carefully timed translation of stored mRNAs in transcriptionally silent cells; however, the molecular mechanisms that determine when individual mRNAs will be translated are not well understood. A large part of the problem results from our having no reliable method of purifying sub-populations of spermatids at different stages of maturation for molecular analysis. The proposed study aims to resolve this deficiency. The applicant proposes to develop a method that uses transgenic mice that expresses the jellyfish green fluorescent protein (GFP) in post- meiotic male germ cells, in combination with fluorescence-activated cell sorting (FACS), to purify homogeneous sub-populations of staged spermatids. The goals of this project are: 1) Isolate at least 20 spermatid sub- populations by FACS. 2) Evaluate the purity and stage of spermatids represented in each sub-population based on morphological, ultrastructural, and molecular criteria; select 10 homogeneous sub-populations representing different stages spanning all of spermiogenesis. 3) Purify mRNP particle- associated mRNA (stored information) and polysome-associated mRNA (active information) from each of these homogeneous sub-populations, prepare a cDNA library from each mRNA sample, and evaluate each by RT-PCR. This work will establish a reliable protocol for sorting spermatids and will yield ordered libraries of most or all of the active and stored spermiogenic information. Our long-term goals are to use this technology to catalog and characterize most or all spermiogenic mRNAs and to determine when each one is used during sperm maturation. In the current submission, the applicant is requesting two years of R03 funding to rigorously establish the technology on which this research, as well as countless other molecular investigations on spermiogenesis, can be founded.