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  • ss-siRNAs allele selectively inhibit ataxin-3 expression: multiple mechanisms for an alternative gene silencing strategy. 23935115

    Single-stranded silencing RNAs (ss-siRNAs) provide an alternative approach to gene silencing. ss-siRNAs combine the simplicity and favorable biodistribution of antisense oligonucleotides with robust silencing through RNA interference (RNAi). Previous studies reported potent and allele-selective inhibition of human huntingtin expression by ss-siRNAs that target the expanded CAG repeats within the mutant allele. Mutant ataxin-3, the genetic cause of Machado-Joseph Disease, also contains an expanded CAG repeat. We demonstrate here that ss-siRNAs are allele-selective inhibitors of ataxin-3 expression and then redesign ss-siRNAs to optimize their selectivity. We find that both RNAi-related and non-RNAi-related mechanisms affect gene expression by either blocking translation or affecting alternative splicing. These results have four broad implications: (i) ss-siRNAs will not always behave similarly to analogous RNA duplexes; (ii) the sequences surrounding CAG repeats affect allele-selectivity of anti-CAG oligonucleotides; (iii) ss-siRNAs can function through multiple mechanisms and; and (iv) it is possible to use chemical modification to optimize ss-siRNA properties and improve their potential for drug discovery.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • ZNF198 stabilizes the LSD1-CoREST-HDAC1 complex on chromatin through its MYM-type zinc fingers. 18806873

    Histone modifications in chromatin regulate gene expression. A transcriptional co-repressor complex containing LSD1-CoREST-HDAC1 (termed LCH hereafter for simplicity) represses transcription by coordinately removing histone modifications associated with transcriptional activation. RE1-silencing transcription factor (REST) recruits LCH to the promoters of neuron-specific genes, thereby silencing their transcription in non-neuronal tissues. ZNF198 is a member of a family of MYM-type zinc finger proteins that associate with LCH. Here, we show that ZNF198-like proteins are required for the repression of E-cadherin (a gene known to be repressed by LSD1), but not REST-responsive genes. ZNF198 binds preferentially to the intact LCH ternary complex, but not its individual subunits. ZNF198- and REST-binding to the LCH complex are mutually exclusive. ZNF198 associates with chromatin independently of LCH. Furthermore, modification of HDAC1 by small ubiquitin-like modifier (SUMO) in vitro weakens its interaction with CoREST whereas sumoylation of HDAC1 stimulates its binding to ZNF198. Finally, we mapped the LCH- and HDAC1-SUMO-binding domains of ZNF198 to tandem repeats of MYM-type zinc fingers. Therefore, our results suggest that ZNF198, through its multiple protein-protein interaction interfaces, helps to maintain the intact LCH complex on specific, non-REST-responsive promoters and may also prevent SUMO-dependent dissociation of HDAC1.
    Document Type:
    Reference
    Product Catalog Number:
    07-455
    Product Catalog Name:
    Anti-CoREST Antibody

  • Analysis of gene function in the retina. 18370205

    The Retina is a good model system for studies of neural development and disease because of its simplicity and accessibility. To analyze gene function rapidly and conveniently, we developed an electroporation technique in mice and rats for use in vivo and in vitro. The efficiency of electroporation into the neonatal retina is quite good, and transgene expression persists for more than a month. With this technique, various types of DNA constructs, including RNA interference (RNAi) vectors, are readily introduced into the retina without DNA size limitation. In addition, more than two different DNA constructs can be introduced into the retina at once, with very high cotransfection efficiency. In vivo and in vitro electroporation will provide a powerful method to analyze the molecular mechanisms of retinal development and disease.
    Document Type:
    Reference
    Product Catalog Number:
    AB5405
    Product Catalog Name:
    Anti-Opsin Antibody, Red/Green

  • Single-stranded RNAs use RNAi to potently and allele-selectively inhibit mutant huntingtin expression. 22939619

    Mutant huntingtin (HTT) protein causes Huntington disease (HD), an incurable neurological disorder. Silencing mutant HTT using nucleic acids would eliminate the root cause of HD. Developing nucleic acid drugs is challenging, and an ideal clinical approach to gene silencing would combine the simplicity of single-stranded antisense oligonucleotides with the efficiency of RNAi. Here, we describe RNAi by single-stranded siRNAs (ss-siRNAs). ss-siRNAs are potent (greater than 100-fold more than unmodified RNA) and allele-selective (greater than 30-fold) inhibitors of mutant HTT expression in cells derived from HD patients. Strategic placement of mismatched bases mimics micro-RNA recognition and optimizes discrimination between mutant and wild-type alleles. ss-siRNAs require Argonaute protein and function through the RNAi pathway. Intraventricular infusion of ss-siRNA produced selective silencing of the mutant HTT allele throughout the brain in a mouse HD model. These data demonstrate that chemically modified ss-siRNAs function through the RNAi pathway and provide allele-selective compounds for clinical development.
    Document Type:
    Reference
    Product Catalog Number:
    12-371
    Product Catalog Name:
    Normal Mouse IgG

  • Drosophila RB proteins repress differentiation-specific genes via two different mechanisms. 20176807

    The RB and E2F proteins play important roles in the regulation of cell division, cell death, and development by controlling the expression of genes involved in these processes. The mechanisms of repression by the retinoblastoma protein (pRB) have been extensively studied at cell cycle-regulated promoters. However, little is known about developmentally regulated E2F/RB genes. Here, we have taken advantage of the simplicity of the E2F/RB pathway in flies to inspect the regulation of differentiation-specific target genes. These genes are repressed by dE2F2/RBF and a recently identified RB-containing complex, dREAM/MMB, in a cell type- and cell cycle-independent manner. Our studies indicate that the mechanism of repression differs from that of cell cycle-regulated genes. We find that two different activities are involved in their regulation and that in proliferating cells, both are required to maintain repression. First, dE2F2/RBF and dREAM/MMB employ histone deacetylase (HDAC) activities at promoter regions. Remarkably, we have also uncovered an unconventional mechanism of repression by the Polycomb group (PcG) protein Enhancer of zeste [E(Z)], which is involved in silencing of these genes through the dimethylation of histone H3 Lys27 at nucleosomes located downstream of the transcription start sites (TSS).
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • A universal plate format for increased throughput of assays that monitor multiple aminoacyl transfer RNA synthetase activities. 17603003

    Aminoacyl transfer RNA (tRNA) synthetases are intensely studied enzymes because of their importance in the establishment of the genetic code and their connection to disease and medicine. During the advancement of this field, several assays were developed. Despite many innovations, the sensitivity, simplicity, and reliability of the radiometric assays (which were among the first to be developed) have ensured their continued use. Four activities are measured by these assays: active site titration, amino acid activation, aminoacylation, and posttransfer editing (deacylation). In an effort to maintain the advantage of these assays while enhancing throughput, reducing waste, and improving data quality, a universal 96-well filter plate format was developed. This format facilitates the assays for all four of the widely studied activities.
    Document Type:
    Reference
    Product Catalog Number:
    05-500

  • Derivation of mouse embryonic stem cells 17487198

    Here we describe a simple and efficient protocol for derivation of germline chimera-competent mouse embryonic stem cells (mESCs) from embryonic day 3.5 (E3.5) blastocysts. The protocol involves the use of early-passage mouse embryonic fibroblast feeders (MEF) and the alternation of fetal bovine serum- and serum replacement (SR)-containing media. As compared to other available protocols for mESCs derivation, our protocol differs in the combination of commercial availability of all reagents, technical simplicity and high efficiency. mESC lines are derived with approximately 50% efficiency (50 independent mESC lines derived from 96 blastocysts). We believe that this protocol could be a good starting point for (i) setting up the derivation of mESC lines in a laboratory and (ii) incorporating further steps to improve efficiency or adapt the protocol to other applications. The whole process (from blastocyst extraction to the freezing of mESC line) usually takes between 15 and 20 d.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Inorganic and organic phosphate measurements in the nanomolar range. 3578786

    A procedure, based on the complex formation of malachite green with phosphomolybdate under acidic conditions, to measure inorganic orthophosphate in the nanomolar range is described. The addition of polyvinyl alcohol is required to stabilize the dye-phosphomolybdate complex. The advantages of the assay are simplicity, stability of the reagents, and high sensitivity. Due to the high permissible acidity in the assay (0.9 N H2SO4), the method can be adapted easily to measure nanomolar amounts of phosphate, liberated from organic compounds like phosphoproteins and phospholipids after wet digestion.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Protocol for the fast chromatin immunoprecipitation (ChIP) method. 17406230

    Chromatin and transcriptional processes are among the most intensively studied fields of biology today. The introduction of chromatin immunoprecipitations (ChIP) represents a major advancement in this area. This powerful method allows researchers to probe specific protein-DNA interactions in vivo and to estimate the density of proteins at specific sites genome-wide. We have introduced several improvements to the traditional ChIP assay, which simplify the procedure, greatly reducing the time and labor required to complete the assay. The simplicity of the method yields highly reproducible results. Our improvements facilitate the probing of multiple proteins in a single experiment, which allows for the simultaneous monitoring of many genomic events. This method is particularly useful in kinetic studies where multiple samples are processed at the same time. Starting with sheared chromatin, PCR-ready DNA can be isolated from 16-24 ChIP samples in 4-6 h using the fast method.
    Document Type:
    Reference
    Product Catalog Number:
    07-030
    Product Catalog Name:
    Anti-dimethyl-Histone H3 (Lys4) Antibody