PURIFYING HOMOGENEOUS SPERMATID SUB-POPULATIONS

描述(改编自申请人的描述)：生精是一种 基于存储的仔细定时平移的顺序装配工艺 转录沉默细胞中的mRNAs；然而，分子机制 决定何时翻译单个mRNAs的方法不是很好 明白了。这个问题很大程度上是由于我们没有可靠的 不同发育阶段精细胞亚群的纯化方法 用于分子分析的成熟技术。拟议的研究旨在解决这一问题 缺乏症。申请人提议开发一种使用转基因技术的方法 表达水母绿色荧光蛋白(GFP)的小鼠 减数分裂雄性生殖细胞与荧光激活细胞的结合 分选(FACS)，以纯化同种亚群的分期精子细胞。 该项目的目标是：1)分离至少20个精子细胞亚群。 通过流式细胞仪进行检测。2)评价精子细胞的纯度和分期。 以形态、超微结构、 和分子标准；选择10个同质亚群代表 精子发生的不同阶段。3)提纯mRNP颗粒- 相关信使核糖核酸(存储信息)和多聚体相关信使核糖核酸(活性 信息)从这些同质亚群中的每一个中，制备出 文库，用RT-PCR方法对文库进行鉴定。这项工作将 建立一种可靠的精子细胞分选方案，并将产生有序的 大多数或所有活跃的和存储的生精信息的库。 我们的长期目标是使用这项技术来编目和表征 大多数或全部生精RNA，并确定每一种在 精子成熟。在当前提交的文件中，申请人要求提供两个 多年的R03资金，以严格建立这项技术 研究，以及无数其他关于精子发生的分子研究， 可以被建立起来。

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.