A library of BAC-GFP reporter recombinants for gene network analysis
In order to identify the cis-regulatory modules that control gene expression during development, we have undertaken the construction of BAC GFP recombinants that contain the intact upstream, downstream and intronic genomic DNA surrounding a particular gene, e.g. the complete cis-regulatory system. These reporters are able to recapitulate the endogenous gene expression with temporal and spatial specificity when microinjected into fertilized sea urchin eggs. We use homologous recombination to target a cassette containing the GFP coding sequence to the start of transcription. A short description of the method is posted here and a more complete method with representative sequences is posted here. The references from which these methods derive are listed below.
From BAC libraries at the Sea Urchin Genome Resource, recombinants have been constructed from four sea urchins, S. purparatus, S. fransciscanus, L variegatus and A. punctulata as well as the sea star A. miniata. Since the Davidson Laboratory is interested in the regulatory domains that control expression of transcription factors and signaling molecules involved in endomesoderm specification, we have first prepared recombinants for several genes that operate in that gene regulatory network, including the transcription factors Bra, Eve, FoxA, GataC, GataE, GCM, Tbr and Krox, as well as differentiation genes endo16 and sm50 and signaling genes Delta and Wnt8 (see Table). For many of these the expression patterns have been verified against their endogenous counterparts.
These BAC clones are available by request. We will also entertain requests to make additional constructs as time permits. For new preparations, you will need to find the BAC by computer or by screening our libraries. In order to prepare a new recombinant BAC we will need the ID of the BAC clone from our libraries, the sequence of the gene it contains and evidence that you know the position of the gene in the BAC. Please inquire of those below if you have questions. This is a bit complicated to explain here.
Yu D, Ellis HM, Lee EC, Jenkins NA, Copeland NG, Court DL. 2000. An efficient recombination system for chromosome engineering in Escherichia coli. Proc Natl Acad Sci U S A. 97:5978-83.
E-Chiang Lee, Daiguan Yu, J. Martinez de Velasco, Lino Tessarollo, Deborah A. Swing, Donald L. Court, Nancy A. Jenkins, and Neal G. Copeland. 2001. A Highly Efficient Escherichia coli-Based Chromosome Engineering System Adapted for Recombinogenic Targeting and Subcloning of BAC DNA. Genomics 73, 56-65.