MicroRNA-122 (miR-122) is an abundant liver-specific miRNA, implicated in fatty acid and cholesterol metabolism as well as hepatitis C viral replication. Here, we report that a systemically administered 16-nt, unconjugated LNA (locked nucleic acid)-antimiR oligonucleotide complementary to the 5′ end of miR-122 leads to specific, dose-dependent silencing of miR-122 and shows no hepatotoxicity in mice. Antagonism of miR-122 is due to formation of stable heteroduplexes between the LNA-antimiR and miR-122 as detected by northern analysis. Fluorescence in situ hybridization demonstrated uptake of the LNA-antimiR in mouse liver cells, which was accompanied by markedly reduced hybridization signals for mature miR-122 in treated mice. Functional antagonism of miR-122 was inferred from a low cholesterol phenotype and de-repression within 24 h of 199 liver mRNAs showing significant enrichment for miR-122 seed matches in their 3′ UTRs. Expression profiling extended to 3 weeks after the last LNA-antimiR dose revealed that most of the changes in liver gene expression were normalized to saline control levels coinciding with normalized miR-122 and plasma cholesterol levels. Combined, these data suggest that miRNA antagonists comprised of LNA are valuable tools for identifying miRNA targets in vivo and for studying the biological role of miRNAs and miRNA-associated gene-regulatory networks in a physiological context.
For the past 15–20 years, the intracellular delivery and silencing activity of oligodeoxynucleotides have been essentially completely dependent on the use of a delivery technology (e.g. lipofection). We have developed a method (called ‘gymnosis’) that does not require the use of any transfection reagent or any additives to serum whatsoever, but rather takes advantage of the normal growth properties of cells in tissue culture in order to promote productive oligonucleotide uptake. This robust method permits the sequence-specific silencing of multiple targets in a large number of cell types in tissue culture, both at the protein and mRNA level, at concentrations in the low micromolar range. Optimum results were obtained with locked nucleic acid (LNA) phosphorothioate gap-mers. By appropriate manipulation of oligonucleotide dosing, this silencing can be continuously maintained with little or no toxicity for >240 days. High levels of oligonucleotide in the cell nucleus are not a requirement for gene silencing, contrary to long accepted dogma. In addition, gymnotic delivery can efficiently deliver oligonucleotides to suspension cells that are known to be very difficult to transfect. Finally, the pattern of gene silencing of in vitro gymnotically delivered oligonucleotides correlates particularly well with in vivo silencing. The establishment of this link is of particular significance to those in the academic research and drug discovery and development communities.
The potency and specificity of locked nucleic acid (LNA) antisense oligonucleotides was investigated as a function of length and affinity. The oligonucleotides were designed to target apolipoprotein B (apoB) and were investigated both in vitro and in vivo. The high affinity of LNA enabled the design of short antisense oligonucleotides (12- to 13-mers) that possessed high affinity and increased potency both in vitro and in vivo compared to longer oligonucleotides. The short LNA oligonucleotides were more target specific, and they exhibited the same biodistribution and tissue half-life as longer oligonucleotides. Pharmacology studies in both mice and non-human primates were conducted with a 13-mer LNA oligonucleotide against apoB, and the data showed that repeated dosing of the 13-mer at 1–2 mg/kg/week was sufficient to provide a significant and long lasting lowering of non-high-density lipoprotein (non-HDL) cholesterol without increasing serum liver toxicity markers. The data presented here show that oligonucleotide length as a parameter needs to be considered in the design of antisense oligonucleotide and that potent short oligonucleotides with sufficient target affinity can be generated using the LNA chemistry. Conclusively, we present a 13-mer LNA oligonucleotide with therapeutic potential that produce beneficial cholesterol lowering effect in non-human primates.
In 2007, the International Knockout Mouse Consortium (IKMC) made the ambitious promise to generate mutations in virtually every protein-coding gene of the mouse genome in a concerted worldwide action. Now, 5 years later, the IKMC members have developed high-throughput gene trapping and, in particular, gene-targeting pipelines and generated more than 17,400 mutant murine embryonic stem (ES) cell clones and more than 1,700 mutant mouse strains, most of them conditional. A common IKMC web portal (www.knockoutmouse.org) has been established, allowing easy access to this unparalleled biological resource. The IKMC materials considerably enhance functional gene annotation of the mammalian genome and will have a major impact on future biomedical research.
A type of retroviral gene trap vectors has been developed that can induce conditional mutations in most genes expressed in mouse embryonic stem (ES) cells. The vectors rely on directional site-specific recombination systems that can repair and reinduce gene trap mutations when activated in succession. After the gene traps are inserted into the mouse genome, genetic mutations can be produced at a particular time and place in somatic cells. In addition to their conditional features, the vectors create multipurpose alleles amenable to a wide range of postinsertional modifications. Here we have used these directional recombination vectors to assemble the largest library of ES cell lines with conditional mutations in single genes yet assembled, presently totaling 1,000 unique genes. The trapped ES cell lines, which can be ordered from the German Gene Trap Consortium, are freely available to the scientific community.conditional mutagenesis ͉ ES cells ͉ gene trapping ͉ site-specific recombination ͉ insertional mutagenesis
This introductory overview shows that cold, in particular frost, stresses a plant in manifold ways and that the plant's response, being injurious or adaptive, must be considered a syndrome rather than a single reaction. In the course of the year perennial plants of the temperate climate zones undergo frost hardening in autumn and dehardening in spring. Using Scots pine (Pinus sylvestris L.) as a model plant the environmental signals inducing frost hardening and dehardening, respectively, were investigated. Over 2 years the changes in frost resistance of Scots pine needles were recorded together with the annual courses of day-length and ambient temperature. Both act as environmental signals for frost hardening and dehardening. Climate chamber experiments showed that short day-length as a signal triggering frost hardening could be replaced by irradiation with far red light, while red light inhibited hardening. The involvement of phytochrome as a signal receptor could be corroborated by respective night-break experiments. More rapid frost hardening than by short day or far red treatment was achieved by applying a short period (6 h) of mild frost which did not exceed the plant's cold resistance. Both types of signals were independently effective but the rates of frost hardening were not additive. The maximal rate of hardening was - 0.93 degrees C per day and frost tolerance of less than< - 72 degrees C was achieved. For dehardening, temperature was an even more effective signal than day-length.
microRNA-155 (miR-155) has been implicated as a central regulator of the immune system, but its function during acute inflammatory responses is still poorly understood. Here we show that exposure of cultured macrophages and mice to lipopolysaccharide (LPS) leads to up-regulation of miR-155 and that the transcription factor c/ebp Beta is a direct target of miR-155. Interestingly, expression profiling of LPS-stimulated macrophages combined with overexpression and silencing of miR-155 in murine macrophages and human monocytic cells uncovered marked changes in the expression of granulocyte colony-stimulating factor (G-CSF), a central regulator of granulopoiesis during inflammatory responses. Consistent with these data, we show that silencing of miR-155 in LPS-treated mice by systemically administered LNA-antimiR results in derepression of the c/ebp Beta isoforms and down-regulation of G-CSF expression in mouse splenocytes. Finally, we report for the first time on miR-155 silencing in vivo in a mouse inflammation model, which underscores the potential of miR-155 antagonists in the development of novel therapeutics for treatment of chronic inflammatory diseases.
The study of genetic disease mechanisms relies mostly on targeted mouse mutants that are derived from engineered embryonic stem (ES) cells. Nevertheless, the establishment of mutant ES cells is laborious and time-consuming, restricting the study of the increasing number of human disease mutations discovered by highthroughput genomic analysis. Here, we present an advanced approach for the production of mouse disease models by microinjection of transcription activator-like effector nucleases (TALENs) and synthetic oligodeoxynucleotides into one-cell embryos. Within 2 d of embryo injection, we created and corrected chocolate missense mutations in the small GTPase RAB38; a regulator of intracellular vesicle trafficking and phenotypic model of HermanskyPudlak syndrome. Because ES cell cultures and targeting vectors are not required, this technology enables instant germline modifications, making heterozygous mutants available within 18 wk. The key features of direct mutagenesis by TALENs and oligodeoxynucleotides, minimal effort and high speed, catalyze the generation of future in vivo models for the study of human disease mechanisms and interventions.gene targeting | mouse genetics | knockout | knockin G ene targeting in embryonic stem (ES) cells is routinely applied to modify the mouse genome and establish the mouse as the most commonly used genetic disease model (1). Nevertheless, the production of targeted mutants is a laborious and time-consuming task that requires the construction of targeting vectors with selection markers, the isolation of mutant ES cells, and the generation of germ-line chimaeras. Moreover, advanced allele design strategies, like the creation of missense mutations, require an additional working step to eliminate selection marker genes from targeted loci (2). Therefore, the establishment of such mutants is presently a low-throughput and long-term procedure, restricting rapid advancements in disease modeling. In contrast, a burst of nucleotide replacements, small deletions and insertions in coding regions that underlie Mendelian or complex human diseases are discovered by high-throughput genomic analysis such as whole-exome sequencing (3-5). The faithful reproduction of such mutations in mouse models will be instrumental in understanding disease mechanisms and to develop therapeutic interventions. This demand is neither covered by available mutagenesis technologies nor by large-scale mouse mutagenesis programs that aim for complete gene inactivation in classical or conditional knockout mice (6). To cope with this challenge, we aimed to create mutations within the shortest time directly in the genome of one-cell mouse embryos, avoiding the time-consuming handling of ES cells, gene-targeting vectors, selection markers, and chimaeras. Our approach is based on transcription activator-like (TAL) effector nucleases (TALENs) to create targeted double-strand breaks that are either sealed by homologous recombination (HR) with mutant, synthetic oligodeoxynucleotides (ODNs) or closed by nonhomologous endj...
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