Browsing by Subject "Oligonucleotides, Antisense"

Now showing 1 - 5 of 5

Open Access
Development of the morpholino gene knockdown technique in Fundulus heteroclitus: a tool for studying molecular mechanisms in an established environmental model.
(Aquat Toxicol, 2008-05-30) Matson, Cole W; Clark, Bryan W; Jenny, Matthew J; Fleming, Carrie R; Hahn, Mark E; Di Giulio, Richard T
A significant challenge in environmental toxicology is that many genetic and genomic tools available in laboratory models are not developed for commonly used environmental models. The Atlantic killifish (Fundulus heteroclitus) is one of the most studied teleost environmental models, yet few genetic or genomic tools have been developed for use in this species. The advancement of genetic and evolutionary toxicology will require that many of the tools developed in laboratory models be transferred into species more applicable to environmental toxicology. Antisense morpholino oligonucleotide (MO) gene knockdown technology has been widely utilized to study development in zebrafish and has been proven to be a powerful tool in toxicological investigations through direct manipulation of molecular pathways. To expand the utility of killifish as an environmental model, MO gene knockdown technology was adapted for use in Fundulus. Morpholino microinjection methods were altered to overcome the significant differences between these two species. Morpholino efficacy and functional duration were evaluated with molecular and phenotypic methods. A cytochrome P450-1A (CYP1A) MO was used to confirm effectiveness of the methodology. For CYP1A MO-injected embryos, a 70% reduction in CYP1A activity, a 86% reduction in total CYP1A protein, a significant increase in beta-naphthoflavone-induced teratogenicity, and estimates of functional duration (50% reduction in activity 10 dpf, and 86% reduction in total protein 12 dpf) conclusively demonstrated that MO technologies can be used effectively in killifish and will likely be just as informative as they have been in zebrafish.
Open Access
Dynamics of Delta/Notch signaling on endomesoderm segregation in the sea urchin embryo.
(Development, 2010-01) Croce, Jenifer C; McClay, David R
Endomesoderm is the common progenitor of endoderm and mesoderm early in the development of many animals. In the sea urchin embryo, the Delta/Notch pathway is necessary for the diversification of this tissue, as are two early transcription factors, Gcm and FoxA, which are expressed in mesoderm and endoderm, respectively. Here, we provide a detailed lineage analysis of the cleavages leading to endomesoderm segregation, and examine the expression patterns and the regulatory relationships of three known regulators of this cell fate dichotomy in the context of the lineages. We observed that endomesoderm segregation first occurs at hatched blastula stage. Prior to this stage, Gcm and FoxA are co-expressed in the same cells, whereas at hatching these genes are detected in two distinct cell populations. Gcm remains expressed in the most vegetal endomesoderm descendant cells, while FoxA is downregulated in those cells and activated in the above neighboring cells. Initially, Delta is expressed exclusively in the micromeres, where it is necessary for the most vegetal endomesoderm cell descendants to express Gcm and become mesoderm. Our experiments show a requirement for a continuous Delta input for more than two cleavages (or about 2.5 hours) before Gcm expression continues in those cells independently of further Delta input. Thus, this study provides new insights into the timing mechanisms and the molecular dynamics of endomesoderm segregation during sea urchin embryogenesis and into the mode of action of the Delta/Notch pathway in mediating mesoderm fate.
Open Access
Effects of an antisense oligonucleotide inhibitor of C-reactive protein synthesis on the endotoxin challenge response in healthy human male volunteers.
(J Am Heart Assoc, 2014-07-10) Noveck, Robert; Stroes, Erik SG; Flaim, JoAnn D; Baker, Brenda F; Hughes, Steve; Graham, Mark J; Crooke, Rosanne M; Ridker, Paul M
BACKGROUND: C-reactive protein (CRP) binds to damaged cells, activates the classical complement pathway, is elevated in multiple inflammatory conditions, and provides prognostic information on risk of future atherosclerotic events. It is controversial, however, as to whether inhibiting CRP synthesis would have any direct anti-inflammatory effects in humans. METHODS AND RESULTS: A placebo-controlled study was used to evaluate the effects of ISIS 329993 (ISIS-CRPR x) on the acute-phase response after endotoxin challenge in 30 evaluable subjects. Healthy adult males were randomly allocated to receive 6 injections over a 22-day period of placebo or active therapy with ISIS 329993 at 400- or 600-mg doses. Eligible subjects were subsequently challenged with a bolus of endotoxin (2 ng/kg). Inflammatory and hematological biomarkers were measured before and serially after the challenge. ISIS-CRPR x was well tolerated with no serious adverse events. Median CRP levels increased more than 50-fold from baseline 24 hours after endotoxin challenge in the placebo group. In contrast, the median increase in CRP levels was attenuated by 37% (400 mg) and 69% (600 mg) in subjects pretreated with ISIS-CRPR x (P<0.05 vs. placebo). All other aspects of the acute inflammatory response were similar between treatment groups. CONCLUSION: Pretreatment of subjects with ISIS-CRPR x selectively reduced the endotoxin-induced increase in CRP levels in a dose-dependent manner, without affecting other components of the acute-phase response. These data demonstrate the specificity of antisense oligonucleotides and provide an investigative tool to further define the role of CRP in human pathological conditions.
Open Access
Microbial inactivation of Pseudomonas putida and Pichia pastoris using gene silencing.
(Environ Sci Technol, 2010-05-01) Morse, Thomas O; Morey, Sara J; Gunsch, Claudia K
Antisense deoxyoligonucleotide (ASO) gene silencing was investigated as a potential disinfection tool for industrial and drinking water treatment application. ASOs bind with their reverse complementary mRNA transcripts thereby blocking protein translation. While ASO silencing has mainly been studied in medicine, it may be useful for modulating gene expression and inactivating microorganisms in environmental applications. In this proof of concept work, gene targets were sh ble (zeocin resistance) and todE (catechol-2,3-dioxygenase) in Pichia pastoris and npt (kanamycin resistance) in Pseudomonas putida. A maximum 0.5-fold decrease in P. pastoris cell numbers was obtained following a 120 min incubation with single-stranded DNA (ssDNA) concentrations ranging from 0.2 to 200 nM as compared to the no ssDNA control. In P. putida, a maximum 5.2-fold decrease was obtained after 90 min with 400 nM ssDNA. While the silencing efficiencies varied for the 25 targets tested, these results suggest that protein activity as well as microbial growth can be altered using ASO gene silencing-based tools. If successful, this technology has the potential to eliminate some of the environmental and health issues associated with the use of strong chemical biocides. However, prior to its dissemination, more research is needed to increase silencing efficiency and develop effective delivery methods.
Open Access
The prevalence and regulation of antisense transcripts in Schizosaccharomyces pombe.
(PLoS One, 2010-12-20) Ni, Ting; Tu, Kang; Wang, Zhong; Song, Shen; Wu, Han; Xie, Bin; Scott, Kristin C; Grewal, Shiv I; Gao, Yuan; Zhu, Jun
A strand-specific transcriptome sequencing strategy, directional ligation sequencing or DeLi-seq, was employed to profile antisense transcriptome of Schizosaccharomyces pombe. Under both normal and heat shock conditions, we found that polyadenylated antisense transcripts are broadly expressed while distinct expression patterns were observed for protein-coding and non-coding loci. Dominant antisense expression is enriched in protein-coding genes involved in meiosis or stress response pathways. Detailed analyses further suggest that antisense transcripts are independently regulated with respect to their sense transcripts, and diverse mechanisms might be potentially involved in the biogenesis and degradation of antisense RNAs. Taken together, antisense transcription may have profound impacts on global gene regulation in S. pombe.