BackgroundExosomes, endosome-derived membrane microvesicles, contain specific RNA transcripts that are thought to be involved in cell-cell communication. These RNA transcripts have great potential as disease biomarkers. To characterize exosomal RNA profiles systemically, we performed RNA sequencing analysis using three human plasma samples and evaluated the efficacies of small RNA library preparation protocols from three manufacturers. In all we evaluated 14 libraries (7 replicates).ResultsFrom the 14 size-selected sequencing libraries, we obtained a total of 101.8 million raw single-end reads, an average of about 7.27 million reads per library. Sequence analysis showed that there was a diverse collection of the exosomal RNA species among which microRNAs (miRNAs) were the most abundant, making up over 42.32% of all raw reads and 76.20% of all mappable reads. At the current read depth, 593 miRNAs were detectable. The five most common miRNAs (miR-99a-5p, miR-128, miR-124-3p, miR-22-3p, and miR-99b-5p) collectively accounted for 48.99% of all mappable miRNA sequences. MiRNA target gene enrichment analysis suggested that the highly abundant miRNAs may play an important role in biological functions such as protein phosphorylation, RNA splicing, chromosomal abnormality, and angiogenesis. From the unknown RNA sequences, we predicted 185 potential miRNA candidates. Furthermore, we detected significant fractions of other RNA species including ribosomal RNA (9.16% of all mappable counts), long non-coding RNA (3.36%), piwi-interacting RNA (1.31%), transfer RNA (1.24%), small nuclear RNA (0.18%), and small nucleolar RNA (0.01%); fragments of coding sequence (1.36%), 5′ untranslated region (0.21%), and 3′ untranslated region (0.54%) were also present. In addition to the RNA composition of the libraries, we found that the three tested commercial kits generated a sufficient number of DNA fragments for sequencing but each had significant bias toward capturing specific RNAs.ConclusionsThis study demonstrated that a wide variety of RNA species are embedded in the circulating vesicles. To our knowledge, this is the first report that applied deep sequencing to discover and characterize profiles of plasma-derived exosomal RNAs. Further characterization of these extracellular RNAs in diverse human populations will provide reference profiles and open new doors for the development of blood-based biomarkers for human diseases.

The toolbox of rat genetics currently lacks the ability to introduce site-directed, heritable mutations into the genome to create knockout animals. Using engineered zinc-finger nucleases (ZFNs) designed to target an integrated reporter and two endogenous rat genes, Immunoglobulin M (IgM) and Rab38, we demonstrate that a single injection of DNA or mRNA encoding ZFNs into the one-cell rat embryo leads to a high frequency of animals carrying 25-100% disruption at the target locus. These mutations are faithfully and efficiently transmitted through the germline. Our data demonstrate the feasibility of targeted gene disruption in multiple rat strains within four months time, paving the way to a humanized monoclonal antibody platform and additional human disease models.The laboratory rat is a well-established model for the genetic dissection of human diseaserelated traits (1) despite the fact that targeted modification of its genome is largely intractable. We investigated the application of engineered zinc-finger nucleases (ZFNs;(2)) for the elimination of specific rat gene function and generation of "knockout" rats. ZFNs induce sitespecific, double-strand DNA breaks that can be repaired by the error-prone non-homologous end joining DNA repair pathway to result in a targeted mutation (Fig. 1A). In the fruit fly and zebrafish, direct embryo injection of ZFN-encoding mRNA has been used to generate heritable knockout mutations at specific loci (2).The design and validation of ZFN reagents to target a single-copy Green Fluorescent Protein (GFP) transgene inserted in a rat chromosome and two endogenous rat genes, IgM and

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The laboratory rat (Rattus norvegicus) is an indispensable tool in experimental medicine and drug development, having made inestimable contributions to human health. We report here the genome sequence of the Brown Norway (BN) rat strain. The sequence represents a high-quality 'draft' covering over 90% of the genome. The BN rat sequence is the third complete mammalian genome to be deciphered, and three-way comparisons with the human and mouse genomes resolve details of mammalian evolution. This first comprehensive analysis includes genes and proteins and their relation to human disease, repeated sequences, comparative genome-wide studies of mammalian orthologous chromosomal regions and rearrangement breakpoints, reconstruction of ancestral karyotypes and the events leading to existing species, rates of variation, and lineage-specific and lineage-independent evolutionary events such as expansion of gene families, orthology relations and protein evolution.

Levels of recombination vary among species, among chromosomes within species, and among regions within chromosomes in mammals. This heterogeneity may affect levels of diversity, efficiency of selection, and genome composition, as well as have practical consequences for the genetic mapping of traits. We compared the genetic maps to the genome sequence assemblies of rat, mouse, and human to estimate local recombination rates across these genomes. Humans have greater overall levels of recombination, as well as greater variance. In rat and mouse, the size of the chromosome and proximity to telomere have less effect on local recombination rate than in human. At the chromosome level, rat and mouse X chromosomes have the lowest recombination rates, whereas human chromosome X does not show the same pattern. In all species, local recombination rate is significantly correlated with several sequence variables, including GC%, CpG density, repetitive elements, and the neutral mutation rate, with some pronounced differences between species. Recombination rate in one species is not strongly correlated with the rate in another, when comparing homologous syntenic blocks of the genome. This comparative approach provides additional insight into the causes and consequences of genomic heterogeneity in recombination.

Recent research has emphasized the importance of the metabolic cluster, which includes glucose intolerance, dyslipidemia, and high blood pressure, as a strong predictor of the obesity-related morbidities and premature mortality. Fundamental to this association, commonly referred to as the metabolic syndrome, is the close interaction between abdominal fat patterning, total body adiposity, and insulin resistance. As the initial step in identifying major genetic loci influencing these phenotypes, we performed a genomewide scan by using a 10-centiMorgan map in 2,209 individuals distributed over 507 nuclear Caucasian families. Pedigreebased analysis using a variance components linkage model demonstrated a quantitative trait locus (QTL) on chromosome 3 (3q27) strongly linked to six traits representing these fundamental phenotypes [logarithm of odds (lod) scores ranged from 2.4 to 3.5]. This QTL exhibited possible epistatic interaction with a second QTL on chromosome 17 (17p12) strongly linked to plasma leptin levels (lod ‫؍‬ 5.0). Situated at these epistatic QTLs are candidate genes likely to influence two biologic precursor pathways of the metabolic syndrome. O besity is a common and chronic disorder associated with decreased longevity and increased morbidity from a variety of diseases, including type 2 diabetes mellitus, hypertension, stroke, and coronary heart disease (1). Fat distribution, specifically the pattern known as upper-body, abdominal, or visceral obesity, is a major predictor of the adverse metabolic profile predisposing to these health risks (2). Thus, abdominal-visceral fat size has emerged as a significant precursor of glucose intolerance, hyperinsulinemia, elevated plasma triglycerides, decreased high density lipoprotein-cholesterol, and increased blood pressure (3). Fundamental to this metabolic milieu are close interactions between total body adiposity, abdominalvisceral fat size, and insulin resistance. Reaven (4) provided evidence to suggest that resistance to insulin-stimulated glucose uptake is associated with a series of related metabolic variables, termed ''syndrome X,'' which cluster in the same individual and include glucose intolerance, disturbed plasma lipids, and high blood pressure (4). Because of close similarities of these features with those associated with abdominal obesity, the more collective term metabolic syndrome was introduced (5).The etiology of the abdominal obesity-metabolic syndrome is complex and is thought to involve metabolic, neuroendocrine, and genetic interactions. A metabolic-neuroendocrine cascade has been proposed in which increased free fatty acid flux from the highly lipolytic visceral adipocytes, together with imbalances in sex hormones, could cause the insulin resistance and hyperinsulinemia, with their metabolic consequences (6). Weight gain with preferential deposition of adipocytes in the abdominal-visceral region is considered secondary to adoption of Westernized diet, activity lifestyle, and reactivity to emotional, intellectual, and physical stresses (5...

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The zinc finger transcription factor GLI1, which mediates Sonic hedgehog signaling during development, is expressed in several human cancers, including basal cell carcinoma, medulloblastoma, and sarcomas. We identified 147 genes whose levels of expression were significantly altered in RNA obtained from cells demonstrating a transformed phenotype with stable GLI1 expression or stable Ha-ras expression. Comparison of expression profiles from GLI1-and Ha-ras-expressing cells established a set of genes unique to GLI1-induced cell transformation. Thirty genes were altered by stable GLI1 expression, and 124 genes were changed by stable Ha-ras expression. Seven genes had altered expression levels in both GLI1-and Ha-ras-expressing cells. Genes whose expression was altered by GLI1 included cell cycle genes, cell adhesion genes, signal transduction genes, and genes regulating apoptosis. GLI1 consensus DNA-binding sequences were identified in the 5 regions of cyclin D2, IGFBP-6, osteopontin, and plakoglobin, suggesting that these genes represent immediate downstream targets. Gel shift analysis confirmed the ability of the GLI1 protein to bind these sequences. Up-regulation of cyclin D2 and down-regulation of plakoglobin were demonstrated in GLI1-amplified compared with non-amplified human rhabdomyosarcoma cells. Many of the GLI1 targets with known function identified in this study increase cell proliferation, indicating that GLI1-induced cell transformation occurs through multiple downstream pathways.Important gene hierarchies, in part coding for components of signal transduction pathways, regulate growth and differentiation during development. One such pathway is the Sonic hedgehog-Patched-Gli pathway (1). SHH 1 signaling is critical to the genetic specification of fate of many tissues during early organogenesis including the central nervous system (2, 3), lung (4), prostate (5), bone (6 -8), and muscle (9). SHH signaling is mediated by the GLI family of transcription factors (10). One of these genes, GLI1, has been shown to be a transcriptional activator operating through a C-terminal VP-16-like acidic helical domain (11). GLI1 transforms cells in culture, and its expression is associated with significant human cancers including basal cell carcinoma (12), medulloblastoma (13), and sarcomas (14). Few downstream targets of GLI1 are known, which precludes a clear understanding of its action in carcinogenesis. Genetic evidence suggests that PTCH and Wnt genes are downstream targets of GLI1 (15), and biochemical evidence has established HNF-3␤ (Hepatocyte Nuclear Factor-3␤) as a target of GLI1 during development (16).Microarray technology has provided a methodology to study the expression of thousands of genes simultaneously and has been used in many important settings (17). Among these is the dissection of signal transduction pathways. To identify unique downstream targets of GLI1, we have utilized a cell transformation phenotype as a selection system for the stable integration and expression of either GLI1 or Ha-ras in RK3...