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Título

Cracking the genome’s second code: Enhancer detection by combined phylogenetic footprinting and transgenic fish and frog embryos

AutorAllende, Miguel L.; Manzanares, Miguel CSIC ORCID; Tena, Juan J. CSIC ORCID; Feijóo, Carmen Gloria; Gómez-Skarmeta, José Luis CSIC ORCID
Palabras claveEnhacer
Transgenic
Regulatory sequences
Iroquois
Tol2
Phylogeny
Xenopus laevis
Zebrafish
Fecha de publicación12-ago-2006
EditorElsevier
CitaciónMethods 39(3): 212-219 (2006)
ResumenGenes involved in vertebrate development are unusually enriched for highly conserved non-coding sequence elements. These regions are readily detected in silico, by genome-wide sequence comparisons between different vertebrates, from mammals to fish (phylogenetic footprinting). It follows that sequence conservation must be the result of positive selection for an essential physiological role. An obvious possibility is that these conserved sequences possess regulatory or structural functions important for gene expression and, thus, an in vivo assay becomes necessary. We have developed a rapid testing system using zebrafish and Xenopus laevis embryos that allows us to assign transcriptional regulatory functions to conserved non-coding sequence elements. The sequences are cloned into a vector containing a minimal promoter and the GFP reporter, and are assayed for their putative cis-regulatory activity in zebrafish or Xenopus transgenic experiments. Vectors used include plasmid DNA and the Tol2 transposon system in fish and X. laevis. We have followed this logic to detect and analyze conserved elements in an intergenic region present in the Iroquois (Irx) gene clusters of zebrafish, Xenopus tropicalis, Fugu rubripes and mouse. We have assayed ∼50 of these conserved elements and shown that the majority behave as modular positive regulatory elements (enhancers) that contribute to specific temporal and spatial domains that are part of the endogenous gene expression pattern. Moreover, comparison of the activity of cognate Irx enhancers from different organisms demonstrates that conservation of sequence is accompanied by in vivo functional conservation across species. Finally, for some of the most conserved elements, we have been able to identify a critical core sequence, essential for correct enhancer function.
Descripción8 páginas, 1 figura.
Versión del editorhttp://dx.doi.org/10.1016/j.ymeth.2005.12.005
URIhttp://hdl.handle.net/10261/41372
DOI10.1016/j.ymeth.2005.12.005
ISSN1046-2023
E-ISSN1095-9130
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