Browsing by Subject "RNA-seq"

Now showing 1 - 7 of 7
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    Evaluation of swine intestinal epithelial responses to luminal stimuli
    (2023-01) Medida, Ramya Lekha
    Gastrointestinal (GI) tract health or ‘gut health’ is synonymous with the overall health and wellbeing of all animals. Gut health is the result of multiple factors, including proper diet, effective digestion and absorption, adequate immune status, and optimal microbiome. The epithelium of GI tract acts as a barrier for the luminal contents and plays an important role in gut health. Understanding the intestinal epithelial responses to various luminal stimuli can help improve overall animal health, by providing insights for better diet design and for the development of alternative disease treatment and prevention strategies. In this dissertation, the effects of micronutrient supplementation and bacterial infection on the intestine and intestinal epithelium responses have been evaluated with L. intracellularis as a case study for bacterial luminal stimuli and dietary zinc (Zn) as a case study for nutrient stimuli.
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    Generation of genome wide linkage maps for a wild potato and RNA-seq analysis of transgene mediated potato defense mechanisms against late blight in the tubers and foliage
    (2013-02) Gao, Liangliang
    Wild potato Solanum bulbocastanum is a rich source of genetic resistance against a variety of pathogens. This project developed molecular tools and expanded biological knowledge useful for the improvement of cultivated potato (S. tuberosum) using genetic resistance from S. bulbocastanum. First, the genome structure of S. bulbocastanum relative to those of its relatives, cultivated potato and tomato (S. lycopersicon) was determined. Second, to facilitate efforts to improve cultivated potato through the introgression and deployment disease resistance derived from S. bulbocastanum, the phenotypic function and molecular mechanisms of one S. bulbocastanum disease resistance gene were explored in cultivated potato foliage and tubers. For determination of genome structure for S. bulbocastanum, Diversity Arrays Technology (DArT) was employed to generate genome wide linkage maps for the species. Employing a pseudo-testcross mapping strategy, 631 DArT markers were integrated into a composite map comprising 12 linkage groups. Our results represent an over ten-fold increase of total marker density compared to previously available genetic maps for the species. Sequencing and alignment of corresponding DArT clones to reference physical maps from tomato and cultivated potato allowed a direct comparison of marker orders between species. Overall, the S. bulbocastanum genetic maps show higher collinearity with reference potato maps than tomato maps, with seven genome regions supporting a closer phylogenetic relationship between potato and S. bulbocastanum than between tomato and S. bulbocastanum. One dominant US cultivar ‘Russet Burbank’(WT; late blight susceptible in foliage and tuber) and its RB (a late blight resistance gene derived from S. bulbocastanum) transgenic line SP2211 (+RB; late blight resistant in foliage and tuber) were compared in both tubers and foliage in their responses to late blight pathogen attack using an RNA-seq approach. In the tubers, a total of 483 million paired end Illumina RNA-seq reads were generated, representing the transcription of 29,319 potato genes. Differentially expressed genes, gene groups and ontology bins that exhibited differences between the WT and +RB lines were identified. P. infestans transcripts, including those of known effectors, were also identified. Faster and stronger activation of defense related genes, gene groups and ontology bins correlated with successful tuber resistance against P. infestans. Our results suggest that the hypersensitive response is likely a general form of resistance against the hemibiotrophic P. infestans—even in potato tubers, organs that develop below ground. In the foliage, a total of 515 million paired end RNA-seq reads were generated, representing the transcription of 29,970 genes. We compared the differences and similarities of responses to P. infestans in potato foliage and tubers. Differentially expressed genes, gene groups and ontology bins were identified to show similarities and differences in foliage and tuber defense mechanisms. Our results suggest that disease resistance gene dosage and shared biochemical pathways contribute to RB-mediated incompatible potato-P. infestans interactions in both the foliage and tubers.
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    Hepatotoxic and Immunomodulatory Transcriptome Responses to Aflatoxin B1 in the Turkey (Meleagris gallopavo)
    (2015-05) Monson, Melissa
    Hepatoxicity and immunotoxicity from dietary exposure to aflatoxin B1 (AFB1) adversely affect poultry health and production. Domestic turkeys (Meleagris gallopavo) are especially sensitive to AFB1 since they have a deficiency in glutathione-mediated detoxification of the reactive AFB1 intermediate. Changes in gene expression can be used to characterize the molecular mechanisms of toxicity; transcriptome analysis allows investigation of differential expression at the genome-wide level. In this research, Illumina RNA-sequencing (RNA-seq) was used to examine transcriptome responses to AFB1 exposure in the turkey. As the liver is the primary site of AFB1 activation and toxicity, the effects of dietary AFB1 on the domestic turkey liver transcriptome was first investigated by sequencing 4 pooled libraries representing 3 individuals for each of 4 treatment groups. As detailed in Chapter 2, predicted transcripts were de novo assembled and differential expression analysis identified significant effects on transcripts from genes involved in apoptosis, cell cycle regulation and lipid metabolism (like E3 ubiquitin-protein ligase Mdm2 and lipoprotein lipase). In Chapter 3, RNA-seq and de novo transcriptome assembly were performed on 3 individual spleen samples per treatment group (n = 12) collected from the same AFB1 challenge trial. Significant down-regulation of antimicrobial genes (like beta-defensin 1) and up-regulation of cytotoxic and antigen presentation genes (such as granzyme A) were observed after AFB1 treatment. Another aspect of these studies was to evaluate the ability of a Lactobacillus-based dietary probiotic to reduce AFB1-effects in the liver and spleen. Addition of probiotics during AFB1 exposure modulated expression in both tissues. Many AFB1-induced expression changes were not mitigated in liver, and although probiotics had some amelioratory effects in the spleen, they were also broadly suppressive of immune genes. Multiple genes impacted in the spleen transcriptome belonged to the Major Histocompatibility Complex (MHC), a region of the genome with genes essential to immune functions. The functions and expression patterns of many of the genes located in the turkey MHC have not been characterized. A single-gene investigation (Chapter 4) characterized expression patterns of 29 MHC genes in the domestic turkey and provided first evidence for expression of B-butyrophilin 2 in muscle tissue. Understanding these expression profiles will help determine MHC gene functions and provide background for expression changes from immunological challenges like AFB1. Unlike the domestic turkey, Eastern wild turkeys (M. g. silvestris) are more resistant to aflatoxicosis due at least in part to their ability to detoxify AFB1. In Chapter 5, an in ovo exposure model was utilized to directly compare the effects of AFB1 exposure in domestic and wild turkey embryos. Embryonic exposure has applicability to poultry production since AFB1 can be maternally transferred into eggs. RNA-seq datasets from embryonic liver tissue in domestic (n = 24) and wild (n =15) turkeys were mapped to a MAKER turkey gene set. Differential expression and pathway analysis identified conserved effects on cell cycle regulators (like E3 ubiquitin-protein ligase Mdm2) and variable effects in genes encoding detoxifying and anti-oxidant enzymes (like glutathione S-transferases) in domestic and wild turkeys. Overall, transcriptome analysis identified hepatic and splenic responses to AFB1, evaluated the use of probiotics, directly compared domestic and wild turkeys, and provided gene targets for future investigation of the molecular mechanisms of aflatoxicosis.
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    High-throughput transcriptomic analysis of resource-poor mammalian cell lines for recombinant protein production
    (2013-10) Johnson, Kathryn Christine
    Over the past 30 years, mammalian cell culture has enabled the production of recombinant protein therapeutics for treatment of a broad range of debilitating or life-threatening diseases. Continual improvements in cell and process engineering have facilitated the attainment of once unheard-of product titers, and improvements in molecular analysis techniques and process analytic technologies have been employed with great success for cell culture process characterization. High-throughput transcriptomic analysis tools such as microarrays and next-generation RNA sequencing (RNA-seq) provide access to gene expression information by simultaneously measuring the expression levels of tens of thousands of genes. However, until recently such tools have not been used to their full advantage in mammalian cell culture processes due to limitations in available reference sequences for the industrially important Chinese hamster ovary (CHO) and baby hamster kidney (BHK) cell lines. We employed high-throughput RNA sequencing in several CHO cell lines to identify and interrogate a class of small non-coding RNAs called microRNAs (miRNAs), which mediate post-transcriptional repression of protein-coding genes. We annotated and analyzed the expression and genomic conservation of several hundred of these small RNAs. We also employed RNA sequencing to build a comprehensive reference transcriptome for a recombinant protein-producing BHK cell lines. We utilized the BHK reference sequence to enable analysis of gene expression levels in the BHK cell line and two Syrian hamster tissues. We designed an expression microarray from the BHK sequence and utilized it to analyze the transcriptome profiles of BHK cells at several time points in perfusion culture at manufacturing scale. Implementation of several functional analysis tools revealed a consistent time-dependent change in the transcriptome profile that involved down-regulation of extracellular matrix components and changes to calcium signaling genes. The transcriptomic reference sequences we developed in this research and the detailed studies they have enabled will enhance our ability to understand and further optimize cell culture processes.
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    Isolation and identification of De Novo long noncoding RNAs from mouse myoblasts and embryonic stem cells
    (2013-01) Lee, Catherine Ann Alsager
    Long noncoding RNAs (lncRNAs) are a pervasive class of transcripts whose importance and biological relevance are only beginning to be elucidated. LncRNAs have been detected in nearly every cell type and found to be fundamentally involved in many biological processes; however, studies that characterize lncRNA expression during certain periods of development are largely missing. Here, we demonstrate how a pool of potentially relevant lncRNAs can be identified using a RNA-chromatin immunoprecipitation (RNA-ChIP) technique that pulls down sufficient amount of RNA to send for sequencing. In our initial experiment, we attempted to identify lncRNAs bound to the MyoD protein in myoblast cells; however, the lack of immunoprecipitation-compatible highly specific antibodies against MyoD prevented us from pursuing this project. As an alternative, we successfully identified lncRNAs bound to the histone-modifying complex COMPASS, as well as those bound to the master pluripotency factors Oct4 and Sox2 in mouse embryonic stem cells. This study provides a proof-of-principle to identify lncRNAs potentially involved in chromatin regulation of pluripotency.
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    Repeated morphine exposure activates synaptogenesis and other neuroplasticity-related gene networks in the dorsomedial prefrontal cortex of male and female rats
    (2023-04) Liu, Shirelle
    Opioid abuse is a chronic disorder likely involving stable neuroplastic modifications. While a number of molecules contributing to these changes have been identified, the broader spectrum of genes and gene networks that are affected by repeated opioid administration remain understudied.In this study, Next-Generation RNA-sequencing (RNA-seq) was employed followed by quantitative chromatin immunoprecipitation to investigate changes in gene expression and their regulation in adult male and female rats’ dorsomedial prefrontal cortex (dmPFC) after a regimen of daily injection of morphine (5.0 mg/kg; 10 days). Ingenuity Pathway Analysis (IPA) was used to analyze affected molecular pathways, gene networks, and associated regulatory factors. A complementary behavioral study evaluated the effects of the same morphine injection regimen on locomotor activity, pain sensitivity, and somatic withdrawal signs. Behaviorally, repeated morphine injection induced locomotor hyperactivity and hyperalgesia in both sexes. 90% of differentially expressed genes (DEGs) in morphine-treated rats were upregulated in both males and females, with a 35% overlap between sexes. A substantial number of DEGs play roles in synaptic signaling and neuroplasticity. Chromatin immunoprecipitation revealed enrichment of H3 acetylation, a transcriptionally activating chromatin mark. Although broadly similar, some differences were revealed in the gene ontology networks enriched in females and males. The results cohere with findings from previous studies based on a priori gene selection. This study also reveals novel genes and molecular pathways that are upregulated by repeated morphine exposure, with some common to males and females and others that are sex-specific.
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    Whole Genome Assembly and Annotation of Northern Wild Rice (Zizania palustris L.), a North American Grain
    (2021-07-23) Haas, Matthew W; Kono, Thomas; Macchietto, Marissa; Millas, Reneth; McGilp, Lillian; Shao, Mingqin; Duquette, Jacques; Hirsch, Candice N; Kimball, Jennifer A; jkimball@umn.edu; Kimball, Jennifer A; University of Minnesota Cultivated Wild Rice Breeding and Genetics Lab
    Northern Wild Rice (NWR; Zizania palustris L.) is an aquatic grass native to North America that is notable for its nutritious grain. This is an important species with ecological, cultural, and agricultural significance, specifically in the Great Lakes region of the United States. Using long- and short-range sequencing, Hi-C scaffolding, and RNA-seq data from eight tissues, we generated a whole genome de novo assembly and annotation of NWR. The assembly is 1.29 Gb, highly repetitive (~76.0%), and contains 46,421 protein-coding genes. Comparative analyses revealed conservation of large syntenic blocks with Oryza sativa L., which were used to identify putative seed shattering genes. Estimates of divergence times revealed the Zizania genus diverged from Oryza ~26-30 million years ago (MYA), while NWR and Zizania latifolia diverged from one another ~6-8 MYA. Comparative genomics revealed evidence of a whole genome duplication in NWR ~5.3 MYA after the NWR-Z. latifolia speciation event. This high-quality genome assembly and annotation provides is a valuable resource for comparative genomics in the Oryzeae tribe and provides an important resource for future conservation and breeding efforts of NWR.