We propose to develop a communal resource of BAC GFP expression vectors of all transcription factors expressed significantly in the embryo of the sea urchin, Strongylocentrotus purpuratus, the genomic sequence of which will go on line shortly. Critically, as shown by our laboratory, BAC 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 spatially restricted 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 BAC 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 exon l of each gene, and build and authenticate each construct. GFP expression directed by each BAC recombinant will be analyzed to confirm faithful recapitulation of endogenous gene expression. We will also make BAC 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. ? ? ?
| Smith, Joel; Davidson, Eric H (2009) Regulative recovery in the sea urchin embryo and the stabilizing role of fail-safe gene network wiring. Proc Natl Acad Sci U S A 106:18291-6 |
| Wahl, Mary E; Hahn, Julie; Gora, Kasia et al. (2009) The cis-regulatory system of the tbrain gene: Alternative use of multiple modules to promote skeletogenic expression in the sea urchin embryo. Dev Biol 335:428-41 |
| Smith, Joel; Davidson, Eric H (2008) Gene regulatory network subcircuit controlling a dynamic spatial pattern of signaling in the sea urchin embryo. Proc Natl Acad Sci U S A 105:20089-94 |
| Smith, Joel (2008) A protocol describing the principles of cis-regulatory analysis in the sea urchin. Nat Protoc 3:710-8 |
