Browsing by Subject "RNA interference"
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Item Characterization of AtSUVR3 functions in Arabidopsis thaliana using RNA interference(2009-05-15) Wang, TaoVariability of transgene expression levels resulting from gene silencing is considered as ahindrance to the successful application of plant genetic engineering. Towards alleviatinggene silencing, I decided to screen for novel genes involved in transgene silencing and toinvestigate how these genes regulate plant development. Genes encoding putative chromatinremodeling factors, especially those including a SET domain, were selected as candidatetargets. A bioinformatic analysis of the Arabidopsis SET genes (AtSET) was performed andthese genes were classified into 6 groups based on the domain architecture. RNA interference (RNAi) vectors were constructed for ~ 20 AtSET genes and wereintroduced into both wild type lines and transgenic lines silenced for a GFP reporter gene.Surprisingly, altered developmental phenotypes were only observed for three constructs,raising questions as to the effectiveness of the RNAi approach for the chosen Arabidopsissystem. To assess this situation, I targeted a phytoene desaturase (PDS) gene using the sameRNAi approach. Inactivation of PDS renders plant a readily identifiable phenotype. Whereasthe RNAi penetrance in Arabidopsis can be very high, the expressivity of RNAi in varioustissues and among different plants can vary dramatically. Contradictory to previous reports,I found that there is correlation between transcript level and silencing phenotype. Possiblereasons for this discrepancy are discussed. No apparent correlation between transgene copynumber and RNAi phenotypes was observed. Among the three RNAi constructs that caused an abnormal development inArabidopsis, K-23 which targets SuvR3 has the highest expressivity and could reactivate asilenced GFP locus. SuvR3 RNAi lines were selfed for six generations and were screenedfor morphological phenotypes. Abnormal number of flower organs, loss of viability of malegametophytes, and decreased seedling germination percentage were found in SuvR3 RNAilines. A progressive increase in both severity and frequency of abnormal phenotypes wereseen in subsequent generations, suggesting an epigenetic regulatory mechanism involvedwith SuvR3. Alternative splicing of SuvR3 was also observed in most of Arabidopsis tissues.One of the protein isoforms, SuvR3, lacks 16 amino acids within the highly conserved SETdomain. Possible effects of isoform interaction are proposed.Item Genomic Approaches to Study Innate Immune Response to Salmonella Enteritidis Infection in Chickens(2010-01-14) Chiang, Hsin-ISalmonella enterica serovar Enteritidis (SE) is one of the most common food-borne pathogens that cause human salmonellosis. Contamination of consumed poultry products continues to be a global threat to public health. Genetic resistance using genomic approach provides a promising solution to controlling SE infection in poultry. The mechanism of SE resistance in chickens remains elusive. Three different approaches, microarray techology, gene silencing, and computational gene analysis, have been utilized to study SE-induced transcriptional changes of host immune response in the chicken. A whole genome chicken 44K microarray was used to analyze the transcriptome of heterophils from SE-resistant (line A) and SE-susceptible chickens (line B) with/without in vitro SE stimulation. Many differentially expressed immune-related genes were found in the SE-infected to non-infected comparison, where more immune-related genes were down-regulated in line B than line A. These results suggested a similar Toll-like receptor (TLR) regulatory network might exist in heterophils of both lines, and provided strong candidates for further investigating SE resistance and susceptibility in chickens. In the gene silencing study, small interfering RNAs (siRNA) were used to specifically inhibit the expression of NFkB1 in the chicken HD11 macrophage cell line with SE challenge. Genes related to the NF-kB signaling pathway were selected to examine the effect of NFkB1 inhibition on TLR pathway. With 36% inhibition of NFkB1 expression, the results showed an increased expression of TLR4 and interleukin (IL)-6 following SE challenge and suggested a likely inhibitory regulation of NFkB1 on TLR signal pathway. Finally, two novel chicken C-type lectin-like receptors were identified and annotated to chicken CD69 and CD94/NKG2-like with multiple evidences generated by computational (in-silico) sequence analysis. Both genes located in a region on chicken chromosome 1 that is syntenic to mammalian Nature Killer Receptor Complex (NKC) region, which may have existed before the divergence between mammals and aves. While siRNA lays the foundation of using loss-of-function approach on testifying gene-gene interactions, in-silico analysis aids in gathering information of unknown genes of great interest. Both approaches provide great potential to use for down-stream analysis following microarray study.Item Identifying functions of Down syndrome-related genes using RNA interference in C. elegans(2010-12) Griffith, Allison Mooney; Pierce-Shimomura, Jonathan T.; Marcotte, Edward M.Down syndrome is one of the most common genetic disorders, resulting in a range of neurological and neuromuscular disabilities. Although the presence of specific disabilities varies among individuals with Down syndrome, all individuals with Down syndrome are born with hypotonia (low muscle tone) and over half with congenital heart defects. Later in life, all individuals demonstrate intellectual disabilities to varying degrees, while many also develop early-onset Alzheimer’s disease. While the cause of Down syndrome is known to be a triplication of the 21st chromosome, it is unknown how this extraneous genetic material causes the development of these phenotypes. We have begun research into the biological basis of these disabilities using the tiny nematode, Caenorhabditis elegans as a genetic model. We used the technique RNA interference (RNAi), which allows us to study the in vivo function of genes by knocking down their expression one at a time in a living, behaving animal. We have used this technique to systematically study the in vivo function for genes involved in Down syndrome. To this end, we identified and knocked down C. elegans genes with sequence similarity to 67% of genes on the human 21st chromosome genes. We used these RNAi-treated worms to investigate the neuromuscular function of human chromosome 21 gene equivalents by assaying locomotion and pharyngeal pumping in a blinded screen. We used locomotion as a measure of neurological and neuromuscular function, while we used pharyngeal pumping as a model for cardiac function. We also performed an aldicarb screen to examine the role of some of these genes in the function of the synapse. Our experiments have provided valuable insight into the in vivo function of the vast majority of genes on the human 21st chromosome. This will be vital to identify genes that are potentially involved in eliciting Down syndrome-related phenotypes, laying the groundwork for further studies into the neurobiology of Down Syndrome.Item Reprogramming DNA Methylation in Bovine Cells by Knocking Down DNA Methyltransferase-1 with RNA Interference(2010-01-20) Stroud, ToddEmbryos derived by somatic cell nuclear transfer (SCNT) produce few pregnancies that result in a live, healthy offspring. This has largely been attributed to the aberrant reprogramming of the somatic cell DNA used for cloning. In order to improve the efficiency of cloning there is a great deal of research needed to determine the role of proteins involved in early embryonic reprogramming. In addition, studies are needed to determine effects on somatic and embryonic cell development as a result of altering these proteins. In this study we investigate the use of RNA interference in bovine somatic cells and embryos to knock down the expression of DNA methyltransferase-1 (DNMT1), an enzyme responsible for maintenance methylation in mammalian cells. We designed our experiments to test whether or not knocking down the DNMT1 gene would lead to a decrease in global methylation. It is our hypothesis that using somatic cells with reduced methylation may be advantageous for increasing the efficiency of cloning via somatic cell nuclear transfer. To accomplish this task, we have designed an infectious non-replicating lentiviral vector capable of delivering a gene that produces a short hairpin RNA targeting the mRNA of DNMT1. The construct included a sequence coding for green fluorescent protein (GFP) that will allow us to identify cells expressing the hairpin as well as a region coding for neomycin resistance so we could select for a pure population of transgenic cells to use for analysis. Infecting bovine fetal fibroblast cells with genes encoding shRNAs that target DNMT1 was successful. Quantitative real time PCR analysis of DNMT1 mRNA suggests that our shRNAs are capable of an 80% knockdown. The protein blot of indicates up to 90% knockdown of DNMT1. Cells transduced twice with a high titer virus showed the highest knockdown of both DNMT1 mRNA and the protein. Analysis of immunolabeled cytosine methylaiton showed a global decrease in DNA methylation as a result of the DNMT1 knockdown. However, double transduced cells with a high knockdown percentage of DNMT1 mRNA and protein became hypermethylated. The second experiment was conducted to determine the effect of injecting small interfering RNAs (siRNAs) targeting DNMT1 into oocytes prior to parthenogenic activation. This experiment was designed to give us information on the survivability and epigenetic profile of early embryos with decreased DNMT1. Oocytes injected with siRNA targeting DNMT1 had little development past the 8-cell stage as compared to the sham injected oocytes. This treatment group also had decreased DNA methylation as determined by immunolabeling of methylated cytosine residues.Item RNA Interference-Based Approach to Combat Viral Infections: Vesicular Stomatitis Virus Group Prototype(2012-10-19) Ramirez Carvajal, LisbethVesicular stomatitis virus (VSV) is considered a prototype for studying non-segmented negative-stranded ribonucleic acid (RNA) viruses. Livestock are naturally infected by VSV, causing severe economic impact due to lack of any effective treatment. RNA interference (RNAi)-based therapeutics are promising alternatives to control viral infections. Lentiviral vector systems deliver artificial short hairpin RNA (shRNA) into the genome of cells to activate the RNAi pathway. In this study, an RNAi-based approach to generate cell lines with reduced susceptibility to VSV (Indiana) infection was tested. First, eight shRNAs targeting either the nucleocapsid (N), phosphoprotein (P), or the polymerase (L) viral genes were designed and introduced into cell systems. To test the potency of the shRNAs for silencing the target viral transcripts, semi-quantitative polymerase chain reaction (PCR) analysis of viral N, P, and L transcripts was performed. Then, supernatants from infected groups were evaluated by microtitration and immunoblot. Finally, the effect of VSV genomic variability in the target region of shRNAs was predicted by partial sequencing field and laboratory-adapted strains. Viral transcripts were significantly reduced in cells stably expressing shRNAs targeting the N viral gene (nucleotides 67-97 or 1312-1332; p<0.05) or P gene (nucleotides 1772-1792; p<0.05). Reduction in viral transcripts was not observed by other VSV-shRNAs tested. Reduction of viral transcripts by the N-shRNA (sh-1312) was accompanied by a decrease in viral protein. Also, a reduction in the viral particles shed from cells expressing N-shRNAs (nucleotides 67-97, p<0.05) was noted. The results also showed complementarity of target gene sequences for shRNAs in the sequence from the laboratory-adapted strain and single base substitutions in the corresponding regions from VSV field isolates. However, these mismatches did not occur within the seed region of the shRNAs. In conclusion, partial silencing of viral transcripts by a single shRNA does not block VSIV replication; however, partial impairment of VSIV replication was observed in N-shRNAs expressing cells. During infection, the naturally high level of N gene transcription may have modulated the sh-RNA effect. The combination of the most potent shRNAs identified here into a multiple shRNA vector may result in further reduction of viral replication. These data contribute to ongoing development of effective RNAi-based technologies to combat viral diseases.Item The role of a viral microRNA and RNA interference during viral replication in mammalian cells(2012-12) Seo, Gil Ju; Sullivan, Christopher S.RNA interference (RNAi) is an evolutionarily conserved process that regulates gene expression. Host cells and viruses interact in many ways, including through miRNAs and RNAi. Viral miRNAs are encoded when viruses, specially including the the polyoma and herpes families, are transcribed in the nucleus. Some viral miRNAs function to regulate host or viral gene expression. Most viral miRNAs’ functions are not known, however, in great detail. A miRNA can be encoded late during infection, as it is by SV40, a model polyomavirus. This downregulates early viral gene expression by directing mRNA RISC-mediated cleavage. As more polyomaviruses are discovered that are associated with human disease, it becomes more important to understand their function and to uncover whether these emerging viruses encode miRNAs. The work presented here shows the discovery of several viral miRNAs in human polyomaviruses—JCV, BKV, and MCV. In addition, I found that viral miRNAs have the evolutionarily conserved function of negatively regulating viral early gene transcripts at a late stage in the infection. During viral replication, viruses utilize the miRNA components of RNAi. However, in invertebrate organisms RNAi also actively defends against viral infection. It is still being debated, though, whether RNAi plays an antiviral role in mammalian cells. Should it be true that RNAi is an antiviral response in mammalian cells, then what is predicted by such a scenario is inconsistent with my studies. I have found that RNAi is strongly inhibited in the early stages after viral infection. Studies with a chemical mimic of viral infection (poly I:C) imply that the innate cellular immune response is responsible for this inhibition. I investigated the molecular changes, in response to viral infection, (e.g. poly ADP-ribosylation of Ago2) in the RNA-induced silencing complex (RISC). I determined that the inhibition of RNAi is brought about by components of the innate response. Completion of this study details a previously unknown “cross talk” between RNAi and the host innate immune response in mammalian cells. Furthermore, I found mir-17 family attenuates a subclass of interferon-stimulated genes. An understanding of viral miRNA and RNAi offers a clue as to we can use molecular intervention for viral infections.Item Sexual Development and Meiotic Silencing in Neurospora crassa(2014-07-29) Suesc?n Torres, Ana VictoriaMeiotic silencing refers to the mechanism of silencing genes or chromosomes without a homologous counterpart (unpaired) during meiotic prophase I. Meiotic silencing has been described in several eukaryotes, including humans. Failure to complete meiotic silencing may be detrimental to the organism. There is a necessity for understanding the regulation of the process. Neurospora is a powerful model system to study gene silencing phenomena. Numerous genes have been determined to be involved in meiotic silencing in Neurospora; however, very little is known about the molecular mechanisms underlying the process. To understand the regulation of meiotic silencing, it is required to combine different approaches such as genetics, proteomics and biochemical analyses. There is a need for introducing biochemical approaches to the study of meiotic silencing and other processes occurring during sexual development in N. crassa. However, protein extraction from sexual tissue is challenging due to the mechanical difficulties associated with disruption of sexual structure. I standardized a strategy that optimizes protein extraction from sexual tissue. Using this strategy, I studied protein-protein interactions among components of the meiotic silencing machinery and determine the proteome of sexual development. I identified new protein interactions during meiotic silencing in N. crassa, and established protein-binding partners for the suppressor of meiotic silencing SMS-5. These interacting partners, PAF400 and Pianissimo represent new molecular components involved in the nuclear initial stage of the meiotic silencing mechanism. Interactions between SMS-5, PAF400, and Pianissimo may represent the connection between chromatin remodeling, DNA repair, signaling transduction pathways and meiotic silencing. I describe the experiments and data analyses used to develop a comprehensive proteomics data set and a functional catalogue for N. crassa sexual development. I used a global proteomics approach and comparative protein functional analysis to investigate the potential molecular differences between two stages of sexual development in filamentous fungi. The data show that secondary metabolites biosynthesis and cellulase activity are required in fruiting body maturation. N. crassa functional catalogue of sexual development proteins will serve as a reference tool for further studies related to sexual development not only in N. crassa, but also in other filamentous ascomycetes.