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.
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