Regulatory Genomics: BAC-GFP Library of Control Genes

Summary

Principal Investigator: E H Davidson
Abstract: We propose to develop a communal resource of BAG GFP expression vectors of all transcription factors expressed significantlyin the embryo of the sea urchin, Strongylocentrotus purpuratus, the genomic sequence of which will go on line shortly. Critically, as shown by our laboratory, BAG GFP recombinants can be injected into eggs and with high efficiency, and they express accurately, capturing the entire cis-regulatory systems intact. This greatly accelerates detailed cis-regulatory analyses. These recombinants can also be used to introduce exogenous regulatory gene products in temporally and spatiallyrestricted patterns within the embryo and larva. Both of these applications will greatly enhance analysis of how the genomic control systems which direct expression of regulatory genes prescribe early development. Specifically, we propose to isolate BAG recombinants for all relevant transcription factors (-100-200) in which the gene of interest is centrally located within the clone, and then to use standard homologous recombination techniques to replace the first exon of the endogenous gene with the coding sequence of the GFP reporter. An ongoing project within our laboratory has already established the expression profiles of most of the pertinent genes. We will then employ our methods for high throughput production of BAC-GFP recombinants in exonl of each gene, and build and authenticate each construct. GFP expression directed by each BAG recombinant will be analyzed to confirm faithful recapitulation of endogenous gene expression. We will also make BAG recombinants for any other gene encoding any kind of protein, that scientists in the community may request. All information needed by other investigators to facilitate access and promote the use of these tools will be made available on the Sea Urchin Genome web site that is currently administrated by our laboratory. Numerous letters from outside investigators attesting interest in this facility are included as an Appendix to this application.
Funding Period: 2006-04-01 - 2010-03-31
more information: NIH RePORT

Top Publications

  1. ncbi A protocol describing the principles of cis-regulatory analysis in the sea urchin
    Joel Smith
    Division of Biology, California Institute of Technology, 1200 E California Boulevard, Pasadena, California 91125, USA
    Nat Protoc 3:710-8. 2008
  2. pmc Gene regulatory network subcircuit controlling a dynamic spatial pattern of signaling in the sea urchin embryo
    Joel Smith
    Division of Biology 156 29, California Institute of Technology, Pasadena, CA 91125, USA
    Proc Natl Acad Sci U S A 105:20089-94. 2008
  3. pmc The cis-regulatory system of the tbrain gene: Alternative use of multiple modules to promote skeletogenic expression in the sea urchin embryo
    Mary E Wahl
    Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA
    Dev Biol 335:428-41. 2009
  4. pmc Regulative recovery in the sea urchin embryo and the stabilizing role of fail-safe gene network wiring
    Joel Smith
    Division of Biology 156 29, California Institute of Technology, Pasadena, CA 91125, USA
    Proc Natl Acad Sci U S A 106:18291-6. 2009

Detail Information

Publications4

  1. ncbi A protocol describing the principles of cis-regulatory analysis in the sea urchin
    Joel Smith
    Division of Biology, California Institute of Technology, 1200 E California Boulevard, Pasadena, California 91125, USA
    Nat Protoc 3:710-8. 2008
    ..Although the expected time line varies greatly with the depth of information required, results may be obtained in as little as 4-6 months, but more detailed analyses will require several years to complete...
  2. pmc Gene regulatory network subcircuit controlling a dynamic spatial pattern of signaling in the sea urchin embryo
    Joel Smith
    Division of Biology 156 29, California Institute of Technology, Pasadena, CA 91125, USA
    Proc Natl Acad Sci U S A 105:20089-94. 2008
    ..Thus the specific cis-regulatory linkages of the gene regulatory network encode the coordinated spatial expression of Wnt and Notch signaling as they sweep outward across the vegetal plate of the embryo...
  3. pmc The cis-regulatory system of the tbrain gene: Alternative use of multiple modules to promote skeletogenic expression in the sea urchin embryo
    Mary E Wahl
    Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA
    Dev Biol 335:428-41. 2009
    ..This module also mediates a newly discovered negative Erg input which excludes non-skeletogenic mesodermal expression...
  4. pmc Regulative recovery in the sea urchin embryo and the stabilizing role of fail-safe gene network wiring
    Joel Smith
    Division of Biology 156 29, California Institute of Technology, Pasadena, CA 91125, USA
    Proc Natl Acad Sci U S A 106:18291-6. 2009
    ..Genomically encoded backup control circuitry thus provides the mechanism underlying a specific example of the regulative development for which the sea urchin embryo has long been famous...