Abstract
BackgroundThe 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.ResultsIn 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.ConclusionsWe 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.
Highlights
High-throughput construction of humanized mice to study gene expression Gene choosing for this study was based in part on serial analysis of gene expression (SAGE) profiling of brain regions of therapeutic interest, as well as data mining of both the Allen Mouse Brain Atlas (ABA) [39] and Brain Gene Expression Map (BGEM) [6,40,41,42]
The expression pattern of these human genes was analyzed in this study using mouse models generated through the HuGX method, that is, carrying a single copy of a human bacterial artificial chromosome (BAC) MaxiPromoter reporter construct docked at the Hprt
Human regulatory regions specifying expression in adult brain regions of therapeutic interest are functionally conserved from human to mouse Initially, in choosing the BACs for each gene in this study, and again to understand the relevance of the expression pattern results obtained from the four humanized mouse models, we examined the primary literature and public genomic databases
Summary
The big challenge in human genetics is understanding the 98% of the genome that comprises non-coding DNA. Over the past few decades, geneticists have primarily focused their research on protein-coding DNA sequences, leading to the identification of essentially all genes, the understanding of the molecular function for many of them, as well as the implications of gene mutations in (ENCODE) consortium [2] seeking to catalog regulatory elements in the human genome, and the Pleiades Promoter Project [3] identifying brain-specific regulatory elements using humanized mouse models [4,5] The latter project aimed at refining our understanding of regulatory elements, as well as providing researchers with novel tools for directed gene expression in restricted brain regions [5]. For a BAC to be ideally suited for MaxiPromoter design, it has to span the whole predicted gene sequence plus extensive flanking intergenic sequences, but cannot contain the predicted promoter region of an adjoining gene
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