The arrival over the past ten years of genomic tools for analyzing the transcriptome of key neuroendocrine tissues has provided considerable new insights into the complexities of the hypothalamic-pituitary-gonadal axis. These genomic approaches invariably depend upon acute isolation of cell types of interest from complex mixtures of many cell types using technologies like FACS analysis or Laser Cell Capture Microdissection. These approaches suffer from the need to significantly process the tissue containing the cell type of interest, including enzymatic dissociation of cells, perfusion, fixation, embedding of tissues for sectioning, and a variety of other manipulations that may alter the transcriptome of the cell type of interest. The ability to isolate the actively translated transcriptome from a cell type of interest in the context of a complex tissue, without the need for significant manipulation of the tissue of origin, would represent a significant advance in current genomic technology. In this proposal, we describe a novel strategy to rapidly and efficiently isolate the actively translated transcriptome by combining polysome analysis with mouse Cre- Lox technology and homologous recombination in embryonic stem cells. In this proposal, our goal is to develop lines of mice bearing a silent epitope-tagged allele of candidate ribosomal proteins. When bred to transgenic mice expressing Cre recombinase in specific neuroendocrine cell types, these silent alleles will express the epitope-tagged ribosomal proteins, which will be incorporated into actively translating ribosome particles, including polysomes. Polysomes will be immunoprecipitated using antibodies to the epitope tags and run on gradients to separate mRNAs with differing levels of translational efficiency. Low, medium, and highly translated mRNAs expressed in specific neuroendocrine cell types under varying physiological states can then be analyzed on microarrays followed by Quantitative-PCR. ? ?
|Sanz, Elisenda; Yang, Linghai; Su, Thomas et al. (2009) Cell-type-specific isolation of ribosome-associated mRNA from complex tissues. Proc Natl Acad Sci U S A 106:13939-44|