Abstract:
:Human embryonic kidney (HEK293) cells were stably transduced with a retroviral vector containing an expression cassette for a short-lived green fluorescent protein (d2EGFP) and the neomycin resistance gene (Neor). When Neor HEK293 clones were treated with proteasome inhibitors, lactacystin or MG132, an increase in the constitutive levels of d2EGFP expression was observed. Based on flow cytometry, proteasome inhibitors induced a 5- to 10-fold increase in the fluorescent intensity of d2EGFP in HEK293 cell clones. However, in the presence of proteasome inhibitors, HEK293 clones showed a 4- to 6.5-fold increase in d2EGFP concentration as determined by western blot analysis. Our data suggest that d2EGFP is a useful indicator of proteasome inhibition. Therefore, stable expression of d2EGFP in mammalian cells is potentially useful for high-throughput screening of cDNAs or pharmaceutical drugs that repress proteasome functions in vivo.
journal_name
Biotechniquesjournal_title
BioTechniquesauthors
Andreatta C,Nahreini P,Hovland AR,Kumar B,Edwards-Prasad J,Prasad KNdoi
10.2144/01303dd03subject
Has Abstractpub_date
2001-03-01 00:00:00pages
656-60issue
3eissn
0736-6205issn
1940-9818journal_volume
30pub_type
杂志文章相关文献
BIOTECHNIQUES文献大全abstract::Recombinant retroviruses are a common vehicle to deliver an exogenous gene to a target cell, which makes them a useful tool in the field of gene therapy. A major drawback to using recombinant retroviruses is the low titer achieved, resulting in a limited number of target cells infected and subsequently poor expression...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/00294rr05
更新日期:2000-10-01 00:00:00
abstract::The systematic preparation of synthetic peptide combinatorial libraries (SPCLs), each composed of tens of millions of peptides that can be screened in existing diagnostically or pharmacologically relevant in vitro assay systems, is reviewed. The identification of optimal peptide sequences has been achieved through the...
journal_title:BioTechniques
pub_type: 杂志文章
doi:
更新日期:1992-09-01 00:00:00
abstract::A pipeline has been created for the characterization of Arabidopsis thaliana mutants by generating flanking sequence tags (FSTs) and optimized for economic, high-throughput production. The GABI-Kat collection of T-DNA mutagenized A. thaliana plants was used as a source of independent transgenic lines. The pipeline inc...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/03356st01
更新日期:2003-12-01 00:00:00
abstract::We have developed an automated purification method for dye-terminator-based DNA sequencing products using a magnetic bead approach. This 384-well protocol generates sequence fragments that are essentially free of template DNA, salt, and excess dye-terminator products. In comparison with traditional ethanol precipitati...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/02326st05
更新日期:2002-06-01 00:00:00
abstract::Intrinsically disordered proteins (IDPs) are subject to post-translational modifications. This allows the same polypeptide to be involved in different interaction networks with different consequences, ranging from regulatory signalling networks to the formation of membrane-less organelles. We report a robust method fo...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/btn-2019-0033
更新日期:2019-07-01 00:00:00
abstract::Directed evolution is poised to change small molecule discovery and provide greater access to "drug space." Sarah Webb looks into the evolving drug discovery landscape. ...
journal_title:BioTechniques
pub_type: 新闻
doi:10.2144/000114616
更新日期:2017-12-01 00:00:00
abstract::Methods have been developed to rapidly visualize the size distribution of third complementarity-determining regions (CDR3) in immunoglobulin (Ig) and T-cell receptor (TCR) molecules. DNA fragments spanning the Ig or TCR CDR3 are generated by PCR using primers at fixed positions in the variable and constant segments. T...
journal_title:BioTechniques
pub_type:
doi:10.2144/96201st02
更新日期:1996-01-01 00:00:00
abstract::For a feasible and cost-effective impedance measurement of cellular alterations in real-time, we combined commercially available microelectrode arrays (MEAs), consisting of 60 microelectrodes, with a conventional impedance analyzer. For proof of principle, a breast carcinoma cell line (MCF-7) was cultured on MEAs, and...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/000112254
更新日期:2006-10-01 00:00:00
abstract::Metabolic and electrical coupling through gap junction channels is implicated in cell differentiation, tissue homeostasis, and electrotonic propagation of signals in excitable tissues. The characterization of gating properties of these channels requires electrophysiological recordings at both single- and multiple-chan...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/03345rr03
更新日期:2003-05-01 00:00:00
abstract::The trend toward high-throughput techniques in molecular biology and the explosion of online scientific data threaten to overwhelm the ability of researchers to take full advantage of available information. This problem is particularly severe in the rapidly expanding area of gene expression experiments, for example, t...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/99276bc03
更新日期:1999-12-01 00:00:00
abstract::Rapid identification of viruses is needed to monitor the blood supply for emerging threats. Here we present a method that meets these criteria and allows for the shotgun sequencing of novel, uncultured DNA viruses directly from human blood. This method employs selection based on the physical properties of viruses comb...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/000112019
更新日期:2005-11-01 00:00:00
abstract::We have developed a rapid [3H]colchicine competition-binding scintillation proximity assay (SPA) to evaluate antimitotic compounds that bind to the colchicine-binding site on tubulin. The premise of our assay is that compounds will compete with radiolabeled colchicine for the tubulin-binding domain. Biotin-labeled tub...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/00291rr02
更新日期:2000-07-01 00:00:00
abstract::Large genes present particular cloning difficulties, especially when expressed at relatively low levels. We describe a novel method, termed 3' rapid amplification of cDNA ends (RACE) walking, for the rapid determination of unknown 3' flanking sequence of a large cDNA. The technique is a derivative of the anchored PCR ...
journal_title:BioTechniques
pub_type:
doi:10.2144/03344st04
更新日期:2003-04-01 00:00:00
abstract::A fluorescent in situ DNA hybridization assay employing a biotinylated DNA probe was used to visualize single copies of human immunodeficiency virus (HIV) proviral DNA in the nuclei and metaphase chromosomes of infected cells. In clonal cell lines that contain either one or two copies of proviral DNA, the efficiency o...
journal_title:BioTechniques
pub_type: 杂志文章
doi:
更新日期:1990-08-01 00:00:00
abstract::DNA-binding proteins can be purified by their affinity for probes with specific sequences that are tethered to magnetic beads. A restriction site at the end of the tether allows release of probe with factor still on it. This probe with bound factor can be bandshifted directly to compare to bandshifts in whole extracts...
journal_title:BioTechniques
pub_type:
doi:
更新日期:1994-05-01 00:00:00
abstract::A brief description of the setup of an inexpensive densitometric analysis system is provided. This system uses a software package and scanner that can be obtained for a total of less than $1000 and that can be operated by any Macintosh computer, including a MacPlus. In the event that a scanner is already available, th...
journal_title:BioTechniques
pub_type:
doi:
更新日期:1994-06-01 00:00:00
abstract::We have developed a novel vector pTCS, as a tool for efficient selection of open reading frame (ORF)-containing inserts. In pTCS clones containing an insert with an ORF a downstream marker gene (immE3, conferring resistance to colicin) is activated via translational coupling with the insert, and transformed cells can ...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/000112629
更新日期:2007-12-01 00:00:00
abstract::We investigated the ability of an amphipathic oligopeptide to carry a synthetic dsDNA oligonucleotide inside human cells. The oligonucleotide was designed as a decoy binding site for the transcriptional activator of the methylguanine-DNA methyltransferase (MGMT) gene. The complex oligopeptide and decoy were administer...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/02321dd03
更新日期:2002-01-01 00:00:00
abstract::FoLT (formamide low temperature) PCR is a protocol for amplifying DNA directly from whole blood without any preparative steps. Up to 10% (vol/vol) whole blood can be added directly into the tube containing the PCR mixture. There is no need for transfers, centrifugations, pre-boiling or any preparative step. It involve...
journal_title:BioTechniques
pub_type: 杂志文章
doi:
更新日期:1993-02-01 00:00:00
abstract::We describe a simple and efficient system for epitope mapping by cloning random gene fragments into a specially designed gIIIp-based phage display vector. DNA encoding the antigen of interest is PCR-amplified and partially digested with DNaseI to generate 50-150-bp-long fragments, which are polished with T4 DNA polyme...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/99272st04
更新日期:1999-08-01 00:00:00
abstract::The Drosophila heart has gained considerable traction as a model of cardiac development and physiology. Previously we described a semiautomatic optical heartbeat analysis (SOHA) method for quantifying functional parameters from the fly heart that facilitated its use as an organ system and disease model. Here we presen...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/000114255
更新日期:2015-02-01 00:00:00
abstract::Real-time or quantitative loop-mediated isothermal amplification (qLAMP) is a promising technique for the accurate detection of pathogens in organisms and the environment. Here we present a comparative study of the performance of six fluorescent intercalating dyes-SYTO-9, SYTO-13, SYTO-82, SYBR Green I, SYBR Gold, Eva...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/000114432
更新日期:2016-07-01 00:00:00
abstract::Single-cell genomics (SCG) is a recently developed tool to study the genomes of unculturable bacterial species. SCG relies on multiple-strand displacement amplification (MDA), PCR, and next-generation sequencing (NGS); however, obtaining sufficient amounts of high-quality DNA from samples is a major challenge when per...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/000114389
更新日期:2016-03-01 00:00:00
abstract::Many DNA binding proteins are known to regulate gene expression. When that binding is altered, a disease state can result. A common method for measuring DNA binding, namely electrophoretic mobility shift assay (EMSA) is often used but it is not amenable to rapid screening of many samples. As an alternative method, we ...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/000112222
更新日期:2006-09-01 00:00:00
abstract::High-throughput approaches for gene cloning and expression require the development of new nonstandard tools for molecular biologists and biochemists. We introduce a Web-based tool to design primers specifically for the generation of expression clones for both laboratory-scale and high-throughput projects. The applicat...
journal_title:BioTechniques
pub_type:
doi:10.2144/02336bc03
更新日期:2002-12-01 00:00:00
abstract::Normalization is a critical step in the analysis of microarray gene expression data. For dual-labeled array, traditional normalization methods assume that the majority of genes are non-differentially expressed and that the number of overexpressed genes approximately equals the number of under-expressed genes. However,...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/000113195
更新日期:2009-08-01 00:00:00
abstract::To establish a nonradioactive method for demonstrating HPV DNA in routinely treated smears of the uterine cervix (alcohol fixation, staining according to Papanicolaou, preservation), in situ hybridizations were carried out in HeLa and SiHa cells grown on slides. After detailed investigations, the sensitivity and speci...
journal_title:BioTechniques
pub_type: 杂志文章
doi:
更新日期:1988-11-01 00:00:00
abstract::Identification of antimitotic molecules that affect tubulin dynamics is a multistep procedure. It includes in vitro tubulin polymerization assay, studies of a cell cycle effect, and general cytotoxicity assessment. To simplify this lengthy screening protocol, we have introduced and validated an assay system based on t...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/000112193
更新日期:2006-06-01 00:00:00
abstract::Ditag genome scanning (DGS) uses next-generation DNA sequencing to sequence the ends of ditag fragments produced by restriction enzymes. These sequences are compared to known genome sequences to determine their structure. In order to use DGS for large-scale genome structural studies, we have substantially revised the ...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/000113294
更新日期:2009-11-01 00:00:00
abstract::Immunofluorescence quantification of γH2AX foci is a powerful approach to quantify DNA double-strand breaks induced by cancer therapy or accidental exposure to ionizing radiation. Here we report a modification to the γH2AX immunofluorescence labeling method, whereby cells are stained in-solution before being spotted a...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/000113738
更新日期:2011-09-01 00:00:00