1999 — 2000 |
Amieux, Paul S |
F32Activity Code Description: To provide postdoctoral research training to individuals to broaden their scientific background and extend their potential for research in specified health-related areas. |
Low Affinity Neurotrophin Receptor Signaling @ University of Washington
tissue /cell culture; tumor necrosis factor alpha
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0.958 |
2007 — 2008 |
Amieux, Paul S |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
In Vivo Ribosome Tagging in Neuroendocrine Cell Types @ University of Washington
[unreadable] DESCRIPTION (provided by applicant): 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. [unreadable] [unreadable]
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0.958 |
2008 — 2010 |
Amieux, Paul S |
U54Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These differ from program project in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes, with funding component staff helping to identify appropriate priority needs. |
Gene Array Facility Core @ University of Washington
Analysis, Data; Area; Biological; Body Tissues; Cannot achieve a pregnancy; Cell Line; Cell Lines, Strains; CellLine; Cells; Collaborations; Communities; Complex; Computer Programs; Computer software; Condition; Contraceptive Agents; Contraceptives; Core Facility; Custom; Data Analyses; Data Set; Dataset; Development; Difficulty conceiving; Electromagnetic, Laser; Experimental Designs; Future; Gene Expression; Gene Expression Profile; Gene Products, RNA; Genes; Genital System, Male, Testis; Goals; Gonadal structure; Gonads; Human; Human, General; Hypophysis; Hypophysis Cerebri; Hypothalamic structure; Hypothalamus; Infertility; Institution; Investigators; Laboratories; Lasers; Maintenance; Maintenances; Mammals, Mice; Man (Taxonomy); Man, Modern; Methods and Techniques; Methods, Other; Mice; Microdissection; Minority; Murine; Mus; NICHD; National Institute of Child Health and Human Development; Nervous System, Pituitary; Oligo; Oligonucleotides; PDE; PGA; Phosphodiesterases; Pituitary; Pituitary Gland; Polyribosomes; Polysomes; Population; Process; Programs (PT); Programs [Publication Type]; Prostaglandins A; Quality Control; RNA; RNA, Non-Polyadenylated; Radiation, Laser; Reporting; Research; Research Personnel; Researchers; Ribonucleic Acid; Ribosomes; Science; Site; Software; Systems Analyses; Systems Analysis; Techniques; Technology; Testicles; Testis; Tissues; Training; Universities; Walkers; Washington; Work; cell type; computer program/software; cost; cultured cell line; design; designing; experiment; experimental research; experimental study; gene expression signature; hypothalamic; infertile; member; new technology; phosphoric diester hydrolase; platform-independent; programs; receptor expression; reproductive; reproductive axis; research study; transcriptome; unable to bear children
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0.958 |
2009 — 2012 |
Amieux, Paul S Mcknight, George Stanley |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Ribotag: a Novel Technique to Profile Cell Type Specific Gene Expression and Inv @ University of Washington
DESCRIPTION (provided by applicant): The brain is composed of hundreds of different neuronal subtypes that each have unique jobs to do. One of the most significant long-term goals of neuroscience is to understand how these neuronal subtypes do their job by producing specific proteins, contacting other neurons, and changing in response to physiological and pathological contexts. Unfortunately, techniques to capture the total translated mRNA from a defined subtype of neurons as the organism responds to the environment, hormones, drugs, and other regulators are lacking. This proposal addresses that need with a novel biochemical and genetic technique to express epitope-tagged ribosomal proteins in response to a cell type specific Cre recombinase in mouse brain. The polyribosomes containing the transcribed and translatable mRNAs are isolated by immunological techniques and then the RNA is analyzed by PCR, microarray, and next-generation DNA sequencing. Our specific aims are to: (1) Develop techniques to optimize polyribosome immunoprecipitation from brain using the RiboTag mouse that has already been created with an HA-tagged Rpl22 ribosomal protein under Cre recombinase regulation. (2) Create a new RiboTag mouse line with a Flag-tagged Rpl23a that will respond to Cre recombinase activation. (3) Activate Rpl22-HA and Rpl23a- Flag with specific Cre recombinase transgenics to test the ability of the RiboTag isolation technique to detect neuron specific mRNAs and the changes that occur under in vivo physiological regulation. (4) Examine whether the RiboTag techniques differentiate between translated mRNAs and those that are translationally repressed. These novel mouse strains and biochemical methods should allow neuroscientists to measure changes in gene expression and translational regulation with greater resolution and higher throughput than previously available and speed our understanding of the underlying changes in RNA and protein that determine synaptic plasticity. In order to understand how the brain adapts to a changing environment under physiological circumstances or how specific neuronal populations degenerate or malfunction in disease, we must have robust and widely available techniques to measure gene and protein expression in specific neuronal subtypes. The ribosome tagging (RiboTag) technology we are developing will provide such a tool to address questions about gene expression and mRNA translation during adaptive changes in the brain-changes that occur during memory and learning, substance abuse and addiction, aging, and in response to neurological disorders. PUBLIC HEALTH RELEVANCE: In order to understand how the brain adapts to a changing environment, we must understand how genes are regulated in real time in specific neuronal circuits and nuclei in the brain. If successful, the ribosome tagging technology will provide a new technology to address questions about how genes are regulated during adaptive events in the brain-events such as memory consolidation, adaptation to drugs of abuse, and adaptive events related to post traumatic stress.
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0.958 |