Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “reverse genetics”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 883 records · Page 49Linked to original sources
PubMed2004

Double-stranded RNA mediates homology-dependent gene silencing of gamma-tubulin in the human parasite Entamoeba histolytica.

Approaches that eliminate mRNA are a powerful tool for reverse genetics applications in eukaryotic microbes for which gene replacement techniques have not yet been developed. Here, for the first time, we demonstrate that RNA duplexes efficiently inhibit gene expression when introduced into the human parasite Entamoeba histolytica. Chemically synthesized, small interfering RNA (siRNA) were highly specific and efficient in silencing parasite gamma-tubulin mRNA. Use of specific antibodies revealed that microtubules and gamma-tubulin were intra-nuclear in E. histolytica. The RNAi approach to modulation of gamma-tubulin mRNA resulted in loss of the highly organized microtubule array an observation that correlates with a significant reduction of gamma-tubulin as well as of the specific mRNA. Our results suggest that gamma-tubulin is essential for microtubule nucleation in E. histolytica.

Animals↗
PubMed2006

Ebola virus: unravelling pathogenesis to combat a deadly disease.

Ebola virus (EBOV) causes severe haemorrhagic fever leading to up to 90% lethality. Increasingly frequent outbreaks and the placement of EBOV in the category A list of potential biothreat agents have boosted interest in this virus. Furthermore, development of new technologies (e.g. reverse genetics systems) and extensive studies on Ebola haemorrhagic fever (EHF) in animal models have substantially expanded the knowledge on the pathogenic mechanisms that underlie this disease. Two major factors in EBOV pathogenesis are the impairment of the immune response and vascular dysfunction. Here, we attempt to summarize the current knowledge on EBOV pathogenesis focusing on these two factors and on recent progress in the development of vaccines and potential therapeutics.

Animals↗
PubMed2006

Strategies for developing vaccines against H5N1 influenza A viruses.

Recent outbreaks of highly pathogenic avian influenza A virus (H5N1 subtype) infections in poultry and humans (through direct contact with infected birds) have raised concerns that a new influenza pandemic might occur in the near future. Effective vaccines against H5N1 virus are, therefore, urgently needed. Reverse-genetics-based inactivated vaccines have been prepared according to World Health Organization (WHO) recommendations and are now undergoing clinical evaluation in several countries. Here, we review the current strategies for the development of H5N1 influenza vaccines, and future directions for vaccine development.

Animals↗
PubMed2005

Dynamic response of plant genome to ultraviolet radiation and other genotoxic stresses.

Oligonucleotide microarray technology was used to identify genes, which are responding after exposure to UV-C radiation and to other agents causing genotoxic stress. The effect of these conditions on recombinational DNA repair was monitored in parallel. Global changes in gene expression were investigated in Arabidopsis wild-type plants challenged with UV-C, bleomycin, another abiotic agent and xylanase, a biotic factor, all leading to elevated homologous recombination frequencies. The comparison of the expression profile of each treatment allowed defining genes specifically involved in the dynamic response to UV. In the future, the potential roles of such genes in the different forms of stress recognition, signal transduction, and their roles in DNA repair processes will be assessed by using reverse genetic tools available for Arabidopsis thaliana.

Arabidopsis↗
PubMed2004

Lipid species profiling: a high-throughput approach to identify lipid compositional changes and determine the function of genes involved in lipid metabolism and signaling.

The development of electrospray ionization mass spectrometry has provided the foundation for the development of strategies to identify and quantify complex lipids from unfractionated extracts of small biological samples. In the 1990s, the feasibility of detailed lipid profiling was demonstrated; in the past two years, analytical strategies have been extended to include classes of lipids that are unique to plants. High-throughput lipid profiling by electrospray ionization tandem mass spectrometry, in combination with forward- or reverse-genetics approaches, has recently been utilized to identify lipid metabolic pathways that are involved in plant development and stress responses, to specify the roles of particular genes and enzymes in plant responses to environmental cues, to determine the lipid species that serve as the substrates and products of specific enzymes, and to identify lipid-metabolizing enzymes that are involved in varied plant processes.

Genes, Plant↗
PubMed2005

Autophagic recycling: lessons from yeast help define the process in plants.

The autophagic engulfment of cytoplasm and organelles and their delivery to the vacuole have long been speculated to play an essential role in bulk protein turnover in plants. Until recently, however, the importance and the mechanism(s) of action of these processes have remained obscure. Aided by the discovery of numerous orthologs of the yeast AUTOPHAGY (ATG) protein system in Arabidopsis, significant advances have been now made in understanding these processes. Both reverse genetic analyses of the Arabidopsis ATG genes and the use of the encoded proteins as cytological markers have confirmed the presence of autophagy in plants and have demonstrated its importance in nutrient recycling, especially during senescence and growth under carbon- or nitrogen-limiting conditions.

Arabidopsis↗
PubMed2006

Endomembrane proton pumps: connecting membrane and vesicle transport.

pH-homeostasis in the endomembrane system requires the activity of proton-pumps. In animals, the progressive acidification of compartments along the endocytic and secretory pathways is critical for protein sorting and vesicle trafficking, and is achieved by the activity of the vacuolar H(+)-ATPase (V-ATPase). Plants have an additional endomembrane pump, the vacuolar H(+)-pyrophosphatase (V-PPase), and previous research was largely focused on the respective functions of the two pumps in secondary active transport across the tonoplast. Recent approaches, including reverse genetics, have not only provided evidence that both enzymes play unique and essential roles but have also highlighted the important functions of the two proton pumps in endocytic and secretory trafficking.

Adenosine Triphosphatases↗
PubMed2004

Recombinant replication-restricted VSV as an expression vector for murine cytokines.

Vesicular stomatitis virus (VSV) is a prototypic non-segmented, negative-strand RNA virus that rapidly and efficiently shuts down the production of host cell-encoded proteins and utilizes the cell's protein production machinery to express high levels of virally encoded proteins. In an effort to take advantage of this characteristic of VSV, we have employed a reverse genetics system to create recombinant forms of VSV encoding a variety of murine cytokines. Previous studies have revealed that cells infected with recombinant VSV that lack expression of the surface glycoprotein (G protein), designated deltaG-VSV, more efficiently express and secrete recombinant proteins than do recombinant "wild-type" VSV. Therefore, murine cytokine-expressing recombinants were produced as deltaG viruses. Propagation of these deltaG viruses in cells that transiently express G protein in vitro results in G-complemented virions that can infect cells, shut down host protein synthesis, and express at high levels each virally encoded protein (including the designated cytokine). We assessed the ability of each deltaG-VSV construct to express recombinant cytokine by infecting BHK cells and then monitoring/measuring the production of the desired cytokine. When possible, the bioactivity of the cytokine products was also measured. The results presented here reveal that large quantities of bioactive cytokines can be produced rapidly and inexpensively using deltaG-VSV as a protein expression system.

Animals↗
PubMed2026

Aphid symbiotic virus engineered for in vivo expression of insecticidal effectors.

Microbial pesticides are eco-friendly alternatives to chemical pesticides. However, few viral pesticides have been developed. Insects harbor diverse symbiotic viruses, which have the potential to be engineered for translational applications in pest control. Here, we engineered Acyrthosiphon pisum virus (APV), a symbiotic virus of the pea aphid, to deliver anti-aphid effectors using reverse genetics technology. A cytomegalovirus (CMV) promoter-driven APV infectious clone was successfully rescued in pea aphids with the assistance of nanocarrier star polymer (SPc). Based on this infectious clone, the protein coding sequence of chymotrypsin inhibitor variant 8 (Chy8) and the double-stranded RNA sequence targeting the aphid clip-domain serine protease (SPLP) were separately assembled into the APV genome to generate APV-Chy8 and APV-dsSPLP infectious clones, respectively. The recombinant APV clones reduced aphid relative survival rates by 34% and 17% by microinjection, respectively. To enhance the transcriptional efficiency, the APV-Chy8 and APV-dsSPLP clones were transcribed in vitro using the T7 promoter. The in vitro-synthesized APV-Chy8 and APV-dsSPLP clones reduced aphid relative survival rates by 48% and 45% by microinjection, respectively. These results demonstrate that engineered APV can deliver cargos and reduce aphid survival under injection-based experimental conditions, highlighting the potential of symbiotic virus-based vectors for delivering insecticidal effectors.

Animals↗
PubMed2005

Metabolism of amyloid-beta peptide and Alzheimer's disease.

The accumulation of amyloid-beta peptide (Abeta), a physiological peptide, in the brain is a triggering event leading to the pathological cascade of Alzheimer's disease (AD) and appears to be caused by an increase in the anabolic activity, as seen in familial AD cases or by a decrease in catabolic activity. Neprilysin is a rate-limiting peptidase involved in the physiological degradation of Abeta in the brain. As demonstrated by reverse genetics studies, disruption of the neprilysin gene causes elevation of endogenous Abeta levels in mouse brain in a gene-dose-dependent manner. Thus, the reduction of neprilysin activity will contribute to Abeta accumulation and consequently to AD development. Evidence that neprilysin in the hippocampus and cerebral cortex is down-regulated with aging and from an early stage of AD development supports a close association of neprilysin with the etiology and pathogenesis of AD. Therefore, the up-regulation of neprilysin represents a promising strategy for therapy and prevention. Recently, somatostatin, which acts via a G-protein-coupled receptor (GPCR), has been identified as a modulator that increases brain neprilysin activity, resulting in a decrease of Abeta levels. Thus, it may be possible to pharmacologically control brain Abeta levels with somatostatin receptor agonists.

Alzheimer Disease↗
PubMed2004

Untangling multi-gene families in plants by integrating proteomics into functional genomics.

The classification and study of gene families is emerging as a constructive tool for fast tracking the elucidation of gene function. A multitude of technologies can be employed to undertake this task including comparative genomics, gene expression studies, sub-cellular localisation studies and proteomic analysis. Here we focus on the growing role of proteomics in untangling gene families in model plant species. Proteomics can specifically identify the products of closely related genes, can determine their abundance, and coupled to affinity chromatography and sub-cellular fractionation studies, it can even provide location within cells and functional assessment of specific proteins. Furthermore global gene expression analysis can then be used to place a specific family member in the context of a cohort of co-expressed genes. In model plants with established reverse genetic resources, such as catalogued T-DNA insertion lines, this gene specific information can also be readily used for a wider assessment of specific protein function or its capacity for compensation through assessing whole plant phenotypes. In combination, these resources can explore partitioning of function between members and assess the level of redundancy within gene families.

Arabidopsis↗
PubMed2006

Jasmonates in arbuscular mycorrhizal interactions.

The mutualistic interaction between plants and arbuscular mycorrhizal (AM) fungi is believed to be regulated from the plant side among other signals by the action of phytohormones. Evidences for this are based mainly on application experiments and determination of phytohormone levels in AM roots by comparison to non-mycorrhizal roots. In case of jasmonates, additional proof is given by reverse genetic approaches, which led to first insights into their putative role in the establishment and functioning of the symbiosis. This review summarizes the current data about phytohormone action in AM roots and the role of jasmonates in particular.

Cyclopentanes↗
PubMed2006

Cloning and characterization of a Nicotiana tabacum methylputrescine oxidase transcript.

The oxidative deamination of N-methylputrescine is an essential step in both pyridine and tropane alkaloid biosynthesis. Reverse genetic approaches have not resulted in the cloning of a methylputrescine oxidase gene (MPO). However, we have used a homology-based approach to clone a full-length tobacco MPO1 cDNA. The MPO1 gene is part of a small multigene family comprised of approximately six members. MPO1-like transcript levels increased in roots that were either deprived of auxin or treated with methyl jasmonic acid. Similar to other known nicotine biosynthetic genes in domesticated tobacco, MPO1-like mRNA levels were lower in roots with the mutant a and b alleles. The MPO1 protein was expressed in bacteria as a recombinant Thioredoxin-His(6)-MPO1 fusion protein. The recombinant MPO1 protein utilized N-methylputrescine more efficiently than other diamines. Therefore, the kinetic properties of the MPO1 enzyme may play an important role in determining the pyridine alkaloid profiles observed in tobacco roots.

Amino Acid Sequence↗
PubMed2004

Bioinformatics: harvesting information for plant and crop science.

Bioinformatics is an integral aspect of plant and crop science research. Developments in data management and analytical software are reviewed with an emphasis on applications in functional genomics. This includes information resources for Arabidopsis and crop species, and tools available for analysis and visualisation of comparative genomic data. Approaches used to explore relationships between plant genes and expressed sequences are compared, including use of ontologies. The impact of bioinformatics in forward and reverse genetics is described, together with the potential from data mining. The role of bioinformatics is explored in the wider context of plant and crop science.

Algorithms↗
PubMed2006

Xenopus as a model system for vertebrate heart development.

The African clawed frog, Xenopus laevis, is a valuable model system for studies of vertebrate heart development. In the following review, we describe a range of embryological and molecular methodologies that are used in Xenopus research and discuss key discoveries relating to heart development that have been made using this model system. We also discuss how the sequence of the Xenopus tropicalis genome provides a valuable tool for identification of orthologous genes and for identification of evolutionarily conserved promoter elements. Finally, both forward and reverse genetic approaches are currently being applied to Xenopus for the study of vertebrate heart development.

Animals↗
PubMed2005

A whole lotta jumpin' goin' on: new transposon tools for vertebrate functional genomics.

Genome sequences of vertebrate model organisms are becoming available, fuelling the next challenging phase of research: annotating all of the genes with functional information. The functional annotation of all of the approximately 35 000 genes in mammals will undoubtedly require complementing the technologies of forward and reverse genetics in mutagenesis screens. Two recent articles report on the construction of retrotransposable elements that efficiently 'jump' in mammalian cells. These new elements hold great promise as useful vectors for insertional mutagenesis screens in the mouse.

Animals↗
PubMed2005

The dual nature of homologous recombination in plants.

Homologous recombination creates covalent linkages between DNA in regions of highly similar or identical sequence. Recent results from several laboratories, many of them based on forward and reverse genetics in Arabidopsis, give insights into the mechanisms of the enzymatic machinery and the involvement of chromatin in somatic and meiotic DNA recombination. Also, signaling pathways and interconnections between repair pathways are being discovered. In addition, recent work shows that biotic and abiotic influences from the environment can dramatically affect plant genomes. The resulting changes in the DNA sequence, exerted at the level of somatic or meiotic tissue, might contribute to evolution.

Arabidopsis↗
PubMed2005

Using worms to better understand how Bacillus thuringiensis kills insects.

Bacillus thuringiensis is widely used as a biological pesticide to control insects that either cause damage to crops or transmit disease. That it can also target the model organism Caenorhabditis elegans has not only provided exciting new insights into how the toxins produced by the bacterium target their victims but also how target insects counter the attack. Modern approaches such as reverse genetics and microarray technology have revealed novel receptors for the toxins and possible signal transduction pathways induced within the host following intoxication. This article will discuss how these findings fit in with current models and how they might influence future studies.

Animals↗