RESUMEN
BACKGROUND: The next big challenge in human genetics is understanding the 98% of the genome that comprises non-coding DNA. Hidden in this DNA are sequences critical for gene regulation, and new experimental strategies are needed to understand the functional role of gene-regulation sequences in health and disease. In this study, we build upon our HuGX ('high-throughput human genes on the X chromosome') strategy to expand our understanding of human gene regulation in vivo. RESULTS: In all, ten human genes known to express in therapeutically important brain regions were chosen for study. For eight of these genes, human bacterial artificial chromosome clones were identified, retrofitted with a reporter, knocked single-copy into the Hprt locus in mouse embryonic stem cells, and mouse strains derived. Five of these human genes expressed in mouse, and all expressed in the adult brain region for which they were chosen. This defined the boundaries of the genomic DNA sufficient for brain expression, and refined our knowledge regarding the complexity of gene regulation. We also characterized for the first time the expression of human MAOA and NR2F2, two genes for which the mouse homologs have been extensively studied in the central nervous system (CNS), and AMOTL1 and NOV, for which roles in CNS have been unclear. CONCLUSIONS: We have demonstrated the use of the HuGX strategy to functionally delineate non-coding-regulatory regions of therapeutically important human brain genes. Our results also show that a careful investigation, using publicly available resources and bioinformatics, can lead to accurate predictions of gene expression.
Asunto(s)
Encéfalo/metabolismo , Cromosomas Artificiales Bacterianos/genética , Cromosomas Humanos X/genética , Secuencias Reguladoras de Ácidos Nucleicos , Células Amacrinas/citología , Células Amacrinas/metabolismo , Angiomotinas , Animales , Factor de Transcripción COUP II/genética , Sistema Nervioso Central/metabolismo , Biología Computacional , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Eliminación de Gen , Regulación de la Expresión Génica , Técnicas de Sustitución del Gen , Sitios Genéticos , Genoma Humano , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Proteínas de la Membrana/genética , Ratones , Monoaminooxidasa/genética , Proteína Hiperexpresada del Nefroblastoma/genética , Células Ganglionares de la Retina/citología , Células Ganglionares de la Retina/metabolismoRESUMEN
The Pleiades Promoter Project integrates genomewide bioinformatics with large-scale knockin mouse production and histological examination of expression patterns to develop MiniPromoters and related tools designed to study and treat the brain by directed gene expression. Genes with brain expression patterns of interest are subjected to bioinformatic analysis to delineate candidate regulatory regions, which are then incorporated into a panel of compact human MiniPromoters to drive expression to brain regions and cell types of interest. Using single-copy, homologous-recombination "knockins" in embryonic stem cells, each MiniPromoter reporter is integrated immediately 5' of the Hprt locus in the mouse genome. MiniPromoter expression profiles are characterized in differentiation assays of the transgenic cells or in mouse brains following transgenic mouse production. Histological examination of adult brains, eyes, and spinal cords for reporter gene activity is coupled to costaining with cell-type-specific markers to define expression. The publicly available Pleiades MiniPromoter Project is a key resource to facilitate research on brain development and therapies.
