Abstract:
:The breakthrough discovery that double-stranded RNA of 21 nucleotides in length (referred to as short or small interfering RNA; siRNA) can trigger sequence-specific gene silencing in mammalian cells has led to the development of a powerful new approach to study gene function (Dillon et al., 2005; Dykxhoorn et al., 2003; Elbashir et al., 2001; Hannon et al., 2004). Effective delivery of siRNA molecules into target cells or tissues is critical for successful RNA interference (RNAi) application. Here, we describe the use of human immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors for delivery of short hairpin RNA (shRNA), a precursor of siRNA, into primary neurons to suppress gene expression. Major advantages of lentiviral vectors are their ability to transduce nondividing cells and to confer long-term expression of transgenes. This chapter covers selection of short hairpin sequences, vector design, production of lentiviral supernatants, transduction of dissociated primary hippocampal neurons, and testing the effectiveness of shRNA-mediated silencing.
journal_name
Methods Enzymoljournal_title
Methods in enzymologyauthors
Janas J,Skowronski J,Van Aelst Ldoi
10.1016/S0076-6879(06)06046-0subject
Has Abstractpub_date
2006-01-01 00:00:00pages
593-605eissn
0076-6879issn
1557-7988pii
S0076-6879(06)06046-0journal_volume
406pub_type
杂志文章abstract::Unfolded protein response (UPR) is a major reaction to intrinsic stress of eukaryotic organisms and is also related to environmental stress reactions. Among yeasts, stress regulation has mainly been investigated in Saccharomyces cerevisiae, while other species with biotechnological or medical interest are less well un...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-385116-1.00010-8
更新日期:2011-01-01 00:00:00
abstract::The current treatment of neurological and psychiatric diseases is far beyond being satisfactory. In addition to highly complex disease mechanisms, the blood-brain barrier (BBB) also remains as a challenge by limiting the delivery of the majority of currently available therapeutics to the central nervous system. Severa...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-391860-4.00013-6
更新日期:2012-01-01 00:00:00
abstract::Metagenomics has become an important tool for the characterization of microorganisms, as it is independent of their enrichment or cultivation in the laboratory. Its application has led to the discovery of metabolisms from widespread, yet uncharacterized organisms such as the ammonia-oxidizing archaea. Different approa...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-386489-5.00013-0
更新日期:2011-01-01 00:00:00
abstract::MicroRNA (miRNA) genes are transcribed into long primary transcripts (pri-miRNAs) that get processed into mature miRNAs of about 22 nt in length by two different ribonuclease (RNase) III enzymes, Drosha and Dicer. Various experimental protocols have been developed and modified for genetic and biochemical analyses for ...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(07)27005-3
更新日期:2007-01-01 00:00:00
abstract::A simple and convenient method is described to determine primary deuterium kinetic isotope effects (1°DKIEs) on reactions where the hydron incorporated into the reaction product is derived from solvent water. The 1°DKIE may be obtained by 1H NMR analyses as the ratio of the yields of H- and D-labeled products from a r...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2017.06.043
更新日期:2017-01-01 00:00:00
abstract::In vivo genome manipulation through site-directed mutagenesis and chromosome rearrangements has been hindered by the difficulty in achieving high frequencies of targeting and the intensive labor required to create altered genomes that do not contain any heterologous sequence. Here we describe our approach, referred to...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(05)09019-1
更新日期:2006-01-01 00:00:00
abstract::Virtually all eukaryotic cell functions and signaling pathways are regulated by protein phosphorylation. The Rad53 kinase plays crucial roles in the DNA damage response in Saccharomyces cerevisiae and is widely used as a surrogate marker for DNA damage checkpoint activation by diverse genotoxic agents. Most currently ...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2016.09.032
更新日期:2017-01-01 00:00:00
abstract::Extracellular thiol/disulfide redox environments are highly regulated in healthy individuals and become oxidized in disease. This oxidation affects the function of cell surface receptors, ion channels, and structural proteins. Downstream signaling due to changes in extracellular redox potential can be studied using a ...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(10)74010-6
更新日期:2010-01-01 00:00:00
abstract::In the yeast Saccharomyces cerevisiae, autophagy contributes to the sustaining of cell viability under starvation conditions, possibly through the supply of amino acids that is generated as a result of the degradation of cytosolic materials. Therefore, cellular viability is one of the best indexes for monitoring the c...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(08)03202-3
更新日期:2008-01-01 00:00:00
abstract::Embryonic stem (ES) cells are cells derived from the inner cell mass of a blastocyst stage embryo. These self-renewing multipotent cells are able to differentiate to the three embryonic germ layers, the endoderm, ectoderm, and mesoderm, and are thus able to produce virtually all cell types. The ES cell capacity to gen...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(06)18015-5
更新日期:2006-01-01 00:00:00
abstract::Precise investigation and manipulation of gene function often require modulation in a controlled and dynamic manner. In this chapter, we describe the methods to apply HIT systems for drug inducible transcriptional activation or simultaneous activation and genome editing in human cells. Together with those for editing,...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2019.02.032
更新日期:2019-01-01 00:00:00
abstract::T lymphocytes express on their surface a heterodimeric αβ receptor, called the T cell receptor (TCR), which recognizes foreign antigens. Unlike antibodies, the recognition requires both an antigenic peptide epitope and a protein encoded by the major histocompatibility complex (MHC). In contrast to conventional antibod...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-396962-0.00008-2
更新日期:2012-01-01 00:00:00
abstract::Sulfatases hydrolyze sulfate esters on a variety of molecules including glycosaminoglycans, sulfoglycolipids, and cytosolic steroids. These enzymes are found in a wide range of organisms with their basic enzymatic mechanisms broadly conserved. In mammals, many of the sulfatases localize in the lysosome and exhibit enz...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(06)16015-2
更新日期:2006-01-01 00:00:00
abstract::Microsatellite markers are quickly becoming the molecular marker of choice for studies of parentage and kinship in animals. In this chapter, we review methods and give protocols for screening potential microsatellite markers, as well as protocols for genotyping individuals with useful markers once they have been ident...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(05)95014-3
更新日期:2005-01-01 00:00:00
abstract::We have described procedures for collecting, processing, and analyzing kinetic data obtained by photobleaching microscopy of GFP-tagged chromatin proteins in nuclei of cultured living cells. These procedures are useful for characterizing the in vivo binding of chromatin proteins to their natural template--unperturbed,...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/s0076-6879(03)75025-3
更新日期:2004-01-01 00:00:00
abstract::We have developed two screening systems for isolating inhibitors that target bacterial two-component signal transduction: (1) a differential growth assay using a temperature-sensitive yycF mutant (CNM2000) of Bacillus subtilis, which is supersensitive to histidine kinase inhibitors, and (2) a high-throughput genetic s...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(06)22019-6
更新日期:2007-01-01 00:00:00
abstract::All cells in nature are covered with a dense and complex array of glycans. The total glycan repertoire expressed on cells, "the cellular glycome," varies at every level of biological organization, and in response to intrinsic and extrinsic stimuli. The cellular glycome is often referred to as the "cell face," which re...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(10)78008-3
更新日期:2010-01-01 00:00:00
abstract::RNA and DNA carry out diverse functions in biology including catalysis, splicing, gene regulation, and storage of genetic information. Interest has grown in understanding how nucleic acids perform such sophisticated functions given their limited molecular repertoire. RNA can fold into diverse shapes that often perturb...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-801122-5.00009-X
更新日期:2014-01-01 00:00:00
abstract::The analysis of a reaction pathway by stopped-flow and chemical quench-flow methods provides the only means of directly measuring the rates of reaction at the active site of an enzyme. The interpretation of data from each technique is similar and involves solution of the differential equations describing the approach ...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/0076-6879(86)34129-6
更新日期:1986-01-01 00:00:00
abstract::Biotinylated molecules are extensively employed in bioanalytics and biotechnology. The currently available assays for quantification of biotin groups suffer from low sensitivity, low accuracy, or provide highly variable responses for different biotin derivatives. We developed a competitive binding assay in which avidi...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2019.10.028
更新日期:2020-01-01 00:00:00
abstract::A procedure for an in vitro signaling assay is described for the MAPK and NFkappaB pathways. The method uses a membrane-cleared lysate that contains all the soluble components required for activating these signaling cascades. The pathways can be activated by variety of molecules, including kinases, G-proteins, and E3 ...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(07)38024-5
更新日期:2008-01-01 00:00:00
abstract::Chloroplast genomes of land plants and algae contain generally between 100 and 150 genes. These genes are involved in plastid gene expression and photosynthesis and in various other tasks. The function of some chloroplast genes is still unknown and some of them appear to be essential for growth and survival. Repressib...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2014.10.035
更新日期:2015-01-01 00:00:00
abstract::Gene regulatory grids (GRGs) encompass the space of all the possible transcription factor (TF)-target gene interactions that regulate gene expression, with gene regulatory networks (GRNs) representing a temporal and spatial manifestation of a portion of the GRG, essential for the specification of gene expression. Thus...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2016.03.003
更新日期:2016-01-01 00:00:00
abstract::Guanine nucleotide exchange factors (GEFs) are essential regulators of the spatiotemporal conditions of small GTP-binding protein (SMG) activation. Their cellular activities combine the biochemical stimulation of GDP/GTP exchange, which leads to the active conformation of the SMG, to the detection of upstream signals ...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(07)38004-X
更新日期:2008-01-01 00:00:00
abstract::The Duffy blood group antigen is a serpentine protein with seven transmembrane domains that is not coupled to G-proteins or other known intracellular effectors. In addition to erythrocytes, it is also expressed in endothelial cells and neurons. In recent years the Duffy antigen has received much attention because of i...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(09)05409-3
更新日期:2009-01-01 00:00:00
abstract::Powerful specialized software is essential for managing, quantifying, and ultimately deriving scientific insight from results of a microarray experiment. We have developed a suite of software applications, known as TM4, to support such gene expression studies. The suite consists of open-source tools for data managemen...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/S0076-6879(06)11009-5
更新日期:2006-01-01 00:00:00
abstract::Dynamin I is a large GTPase enzyme required in membrane constriction and fission during multiple forms of endocytosis. The first method described here is for the rapid purification of native dynamin from peripheral membrane extracts of sheep brain using ammonium sulfate precipitation and affinity purification on recom...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(05)04049-8
更新日期:2005-01-01 00:00:00
abstract::Although the use of monoclonal antibodies to characterize the Ia antigen system is still in its infancy, the usefulness of this approach is clearly apparent. Since xenogeneic immunization can be used to generate these antibodies, the chances are good that a greater variety of Ia types and determinants will be detected...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/0076-6879(83)92010-4
更新日期:1983-01-01 00:00:00
abstract::Nonsense-mediated mRNA decay (NMD) is an evolutionarily conserved mechanism of specific degradation of transcripts with a premature stop codon. NMD eliminates aberrant mRNAs arising from mutations, alternative splicing, and other events in cells. In addition, many normal transcripts undergo NMD. Recent studies demonst...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2016.02.008
更新日期:2016-01-01 00:00:00
abstract::Heterodimeric complexes between individual members of the R7 subfamily of regulators of G-protein signaling proteins and the Gbeta5 isoform of the heterotrimeric G-protein beta subunit family are strongly expressed in the cell nucleus in neurons and brain, as well as in the cytoplasm and plasma membrane. Native and re...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(04)90014-6
更新日期:2004-01-01 00:00:00