CORE--MOLECULAR TECHNIQUES FACILITY

分子技术核心将为使用自动基因分型机的DNA测序提供中央资源，以及为原位杂交分析提供资源。本单元是本计划项目的重要和关键组成部分。我们设计了一个规划良好的设施来分析不同大小的STS，微卫星，限制较大的阶段和粘粒克隆，并执行自动测序，以满足个别研究人员的需求。所有项目调查人员都需要使用ABI Prism基因分型机。原位杂交是研究精子发生过程中调控细胞分化的基因差异表达的理想方法。项目二和项目三建议使用原位杂交来表征与精子发生和睾丸下降有关的新基因。然而，通常用于制备其他哺乳动物器官组织以进行原位杂交的固定剂和技术严重扭曲了精致的睾丸结构，阻碍了识别在生精增殖和分化过程中表达的细胞和阶段特异性基因的尝试。在保留mRNA信号的同时，福尔马林固定和冰冻组织切片扭曲了睾丸组织学，使得无法获得定位基因表达所需的结构信息。在实验室对方法学进行改进的基础上，我们优化了睾丸组织原位杂交的方法。该中心还将为项目调查人员提供原位杂交服务。

The Molecular Technique Core will provide a central resource for DNA sequencing using an automated genotyping machine, as well as a resource for in situ hybridization analysis. This unit represents a significant and critical component of this Program Project. We designed a well planned facility to analyze STSs of various sizes, microsatellites, to restriction map larger phase and cosmid clones and to perform automated sequencing to met the needs of the individual investigators. All Project Investigators need to use the ABI Prism Genotyping Machine. In situ hybridization is an ideal method to determine the differential expression of genes regulating cellular differentiation during spermatogenesis. Projects II and III propose to use in situ hybridization to characterize new genes involved in spermatogenesis and testicular descent. Nevertheless, the fixatives and techniques commonly used to prepare other mammalian organ tissues for in-situ hybridization severely distort the delicate testicular architecture, thwarting attempts to identify cell- and stage-specific genes expressed during spermatogenic proliferation and differentiation. While preserving the mRNA signal, formalin fixation and frozen tissue sectioning distorts testicular histology and renders unobtainable the very architectural information necessary to localize gene expression. Based upon modification of the methodology by our laboratory, we have optimized a method for in situ hybridization for testicular tissue. This Core will also provide in situ hybridization as a service to Project Investigators.