Construction of a bicistronic vector for the co-expression of two genes in Caenorhabditis elegans using a newly identified IRES.

Abstract:

:The nematode Caenorhabditis elegans is an important model animal for biological research. Currently, transgenic C. elegans strains are mainly generated by injecting DNA encoding a gene of interest, in combination with a reporter gene, into the gonad. With this approach, the interpretation of negative results, such as the failure to observe reporter expression, is frequently required. Single, selectable vectors are urgently required. Internal ribosome entry site (IRES) elements are known to bind the eukaryotic ribosomal translation initiation complex and independently promote translation initiation. Bioinformatic analysis predicted an IRES motif upstream of the start codon of the C. elegans Hsp-3 gene. While this sequence has a Y-shaped double-hairpin secondary structure characteristic of IRES elements, it was unclear if it could function as an IRES. In the present study, this predicted Hsp-3 IRES was incorporated into a bicistronic vector driven by the myo-3 promoter, which allowed co-expression of RFP and GFP genes in the muscle tissue of C. elegans and thereby demonstrated that this IRES element is functional. This vector provides a novel, powerful tool for C. elegans research.

journal_name

Biotechniques

journal_title

BioTechniques

authors

Li D,Wang M

doi

10.2144/000113821

subject

Has Abstract

pub_date

2012-03-01 00:00:00

pages

173-6

issue

3

eissn

0736-6205

issn

1940-9818

pii

000113821

journal_volume

52

pub_type

杂志文章
  • Pulse-first heterofusion of cells by electric field pulses and associated loading of macromolecules into mammalian cells.

    abstract::Exposing cells to brief, high-intensity electrical field pulses can lead to the permeabilization of their plasma membranes. This electro-induced permeated state of the cell membrane is reversible and leads to cell fusion; i.e., the electropermeabilized state if fusogenic. The size of cells intended for fusing, however...

    journal_title:BioTechniques

    pub_type: 杂志文章

    doi:

    authors: Rols MP,Dahhou F,Teissié J

    更新日期:1994-10-01 00:00:00

  • Using phiX174 DNA as an exogenous reference for measuring mitochondrial DNA copy number.

    abstract::Quantitative real-time PCR has become a popular method to analyze and quantify changes in the copy number of mitochondrial DNA (mtDNA), and nuclear DNA (nDNA) is often used as an endogenous reference for mtDNA abundance. In our experience, using nDNA as a reference is problematic, due to differences in the extraction ...

    journal_title:BioTechniques

    pub_type: 杂志文章

    doi:10.2144/000113222

    authors: Myers MB,Mittelstaedt RA,Heflich RH

    更新日期:2009-10-01 00:00:00

  • Generation of clonal DNA templates for in vitro transcription without plasmid purification.

    abstract::WE present a rapid procedure based on the polymerase chain reaction for generation of double-stranded DNA templates suitable for in vitro transcription by T3 or T7 RNA polymerase. DNA fragments cloned into a phage promoter vector are amplified together with a flanking promoter to provide functional templates. Extensio...

    journal_title:BioTechniques

    pub_type: 杂志文章

    doi:

    authors: Weier HU,Rosette C

    更新日期:1990-03-01 00:00:00

  • Simplified DGS procedure for large-scale genome structural study.

    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

    authors: Jung YC,Xu J,Chen J,Kim Y,Winchester D,Wang SM

    更新日期:2009-11-01 00:00:00

  • Improved lysis of single bacterial cells by a modified alkaline-thermal shock procedure.

    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

    authors: He J,Du S,Tan X,Arefin A,Han CS

    更新日期:2016-03-01 00:00:00

  • Method for linking a synthesized protein to its mRNA-DNA complex.

    abstract::A nascent protein remains in a complex with its ribosome and mRNA if the stop codon is deleted from the mRNA. In the same manner, mRNA forms a stable complex with DNA if the transcription termination is blocked. In principle, if both mRNA translation and DNA transcription termination are prevented, the protein should ...

    journal_title:BioTechniques

    pub_type: 杂志文章

    doi:10.2144/00294st06

    authors: Liu S,Shivashankar GV,Sano T,Libchaber A

    更新日期:2000-10-01 00:00:00

  • Subtractive hybridization strategy using paramagnetic oligo(dT) beads and PCR.

    abstract::Subtractive hybridization has been widely used for the identification of differentially expressed genes. Here we describe a simple, sensitive strategy of subtractive hybridization that involves binding the driver poly(A)+ RNA pool to paramagnetic Dynabeads Oligo (dT)25. After hybridization with target cDNA, the molecu...

    journal_title:BioTechniques

    pub_type:

    doi:10.2144/19962003413

    authors: Mészáros M,Morton DB

    更新日期:1996-03-01 00:00:00

  • Destabilized green fluorescent protein detects rapid removal of transcription blocks after genotoxic exposure.

    abstract::High stabilities of reporter proteins and their messenger RNAs (mRNAs) interfere with the detection of rapid transient changes in gene expression, such as transcriptional blocks posed by genotoxic DNA lesions. We have modified a green fluorescent protein (GFP) gene within the episomal pMARS vector by addition of a fra...

    journal_title:BioTechniques

    pub_type: 杂志文章

    doi:10.2144/000112479

    authors: Kitsera N,Khobta A,Epe B

    更新日期:2007-08-01 00:00:00

  • Method for discovering novel DNA viruses in blood using viral particle selection and shotgun sequencing.

    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

    authors: Breitbart M,Rohwer F

    更新日期:2005-11-01 00:00:00

  • Nonradioactive assay of FLAG-tagged MAPK using ANTI-FLAG antibody-coated multiwell plates.

    abstract::We have developed a rapid, sensitive, and quantitative 96-well microplate-based nonradioactive immunoprecipitation/kinase assay to evaluate mitogen-activated protein kinase (MAPK) activity. Three quantitative nonradioactive imunoprecipitation/kinase assays of MAPK were demonstrated on a 96-well microplate coated with ...

    journal_title:BioTechniques

    pub_type: 杂志文章

    doi:10.2144/02322pf02

    authors: Zhang L,Uder S,Juehne T,Brizzard B,Song K

    更新日期:2002-02-01 00:00:00

  • Sea urchin embryo as a model organism for the rapid functional screening of tubulin modulators.

    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

    authors: Semenova MN,Kiselyov A,Semenov VV

    更新日期:2006-06-01 00:00:00

  • Rapid purification of synthetic oligonucleotides: a convenient alternative to high-performance liquid chromatography and polyacrylamide gel electrophoresis.

    abstract::A new method has been developed to purify and detritylate milligram amounts of synthetic oligonucleotides. Dimethoxytrityl oligonucleotides from 15 to 100 nucleotides in length are applied in triethylammonium acetate or concentrated ammonium hydroxide to a disposable chromatographic cartridge, the NENSORB PREP Nucleic...

    journal_title:BioTechniques

    pub_type: 杂志文章

    doi:

    authors: Johnson BA,McClain SG,Doran ER,Tice G,Kirsch MA

    更新日期:1990-04-01 00:00:00

  • Computational methods for gene expression-based tumor classification.

    abstract::Gene expression profiles may offer more or additional information than classic morphologic- and histologic-based tumor classification systems. Because the number of tissue samples examined is usually much smaller than the number of genes examined, efficient data reduction and analysis methods are critical. In this rep...

    journal_title:BioTechniques

    pub_type: 杂志文章

    doi:10.2144/00296bc02

    authors: Xiong M,Jin L,Li W,Boerwinkle E

    更新日期:2000-12-01 00:00:00

  • Production of monoclonal antibodies by genetic immunization.

    abstract::Genetic immunization is a simple method for producing polyclonal antibodies in mice. To test if this approach could be used for monoclonal antibody production, biolistic transfection was used to immunize a mouse. High levels of polyclonal antibodies against human growth hormone (hGH) were elicited following three inoc...

    journal_title:BioTechniques

    pub_type:

    doi:

    authors: Barry MA,Barry ME,Johnston SA

    更新日期:1994-04-01 00:00:00

  • A simple method for isolation of DNA from blood clots suited for use in PCR.

    abstract::We describe a rapid, simple and inexpensive method for the isolation of DNA from blood clots suited for use in PCR. Our method is based on the lysing and nuclease-inactivating properties of guanidine thiocyanate together with the nucleic acid-binding properties of silica particles. Isolated DNA can be used for in vitr...

    journal_title:BioTechniques

    pub_type: 杂志文章

    doi:

    authors: Zeillinger R,Schneeberger C,Speiser P,Kury F

    更新日期:1993-02-01 00:00:00

  • A streamlined ribosome profiling protocol for the characterization of microorganisms.

    abstract::Ribosome profiling is a powerful tool for characterizing in vivo protein translation at the genome scale, with multiple applications ranging from detailed molecular mechanisms to systems-level predictive modeling. Though highly effective, this intricate technique has yet to become widely used in the microbial research...

    journal_title:BioTechniques

    pub_type: 杂志文章

    doi:10.2144/000114302

    authors: Latif H,Szubin R,Tan J,Brunk E,Lechner A,Zengler K,Palsson BO

    更新日期:2015-06-01 00:00:00

  • Efficacy of an amphipathic oligopeptide to shuttle and release a cis-acting DNA decoy into human cells.

    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

    authors: Citti L,Rovero P,Colombo MG,Mariani L,Poliseno L,Rainaldi G

    更新日期:2002-01-01 00:00:00

  • TEV protease-mediated cleavage in Drosophila as a tool to analyze protein functions in living organisms.

    abstract::Drosophila provides a powerful experimental system to analyze gene functions in a multi-cellular organism. Here we describe an in vivo method that interferes with the integrity of selected proteins through site-specific cleavage in Drosophila. The technique is based on the highly specific seven-amino-acid recognition ...

    journal_title:BioTechniques

    pub_type: 杂志文章

    doi:10.2144/000112884

    authors: Harder B,Schomburg A,Pflanz R,Küstner K,Gerlach N,Schuh R

    更新日期:2008-05-01 00:00:00

  • A rapid method for site-specific mutagenesis and directional subcloning by using the polymerase chain reaction to generate recombinant circles.

    abstract::Site-specific mutagenesis and directional subcloning were accomplished by using the polymerase chain reaction to generate products that can recombine to form circular DNA. This DNA was transfected into E. coli without phosphorylation of primers, restriction enzyme digestion or ligation. Specifically, the polymerase ch...

    journal_title:BioTechniques

    pub_type: 杂志文章

    doi:

    authors: Jones DH,Howard BH

    更新日期:1990-02-01 00:00:00

  • AMDA 2.13: A major update for automated cross-platform microarray data analysis.

    abstract::Microarray platforms require analytical pipelines with modules for data pre-processing including data normalization, statistical analysis for identification of differentially expressed genes, cluster analysis, and functional annotation. We previously developed the Automated Microarray Data Analysis (AMDA, version 2.3....

    journal_title:BioTechniques

    pub_type: 杂志文章

    doi:10.2144/0000113889

    authors: Kapetis D,Clarelli F,Vitulli F,de Rosbo NK,Beretta O,Foti M,Ricciardi-Castagnoli P,Zolezzi F

    更新日期:2012-07-01 00:00:00

  • Screening for important base identities in the hairpin ribozyme by in vitro selection for cleavage.

    abstract::Random mutagenesis followed by an in vitro selection procedure was shown to be capable of identifying important bases of the hairpin ribozyme for cleavage of an RNA target sequence. The selection scheme enriched the RNA population for those molecules capable of efficient site-specific self-cleavage in the absence of l...

    journal_title:BioTechniques

    pub_type: 杂志文章

    doi:10.2144/98242st05

    authors: Siwkowski A,Humphrey M,De-Young MB,Hampel A

    更新日期:1998-02-01 00:00:00

  • Simplified gene-fragment phage display system for epitope mapping.

    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

    authors: Gupta S,Arora K,Sampath A,Khurana S,Singh SS,Gupta A,Chaudhary VK

    更新日期:1999-08-01 00:00:00

  • Method for isolation of PCR-ready genomic DNA from zebrafish tissues.

    abstract::Here we describe a method for the isolation of PCR-ready genomic DNA from various zebrafish tissues that is based on a previously published murine protocol. The DNA solutions are of sufficient quality to allow PCR detection of transgenes from all commonly used zebrafish tissues. In sperm, transgene amplification was s...

    journal_title:BioTechniques

    pub_type: 杂志文章

    doi:10.2144/000112619

    authors: Meeker ND,Hutchinson SA,Ho L,Trede NS

    更新日期:2007-11-01 00:00:00

  • PART I: FIGHTING CANCER WITH DEADLY BACTERIA.

    abstract::A 200-year-old observation might provide a new way to eliminate tumors by infecting cancer patients with bacteria. Kristie Nybo explores a new approach that could transform cancer treatment. ...

    journal_title:BioTechniques

    pub_type: 新闻

    doi:10.2144/000114624

    authors: Nybo K

    更新日期:2018-01-01 00:00:00

  • Comparison of fluorescent intercalating dyes for quantitative loop-mediated isothermal amplification (qLAMP).

    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

    authors: Oscorbin IP,Belousova EA,Zakabunin AI,Boyarskikh UA,Filipenko ML

    更新日期:2016-07-01 00:00:00

  • A novel vector for positive selection of inserts harboring an open reading frame by translational coupling.

    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

    authors: Ohashi-Kunihiro S,Yohda M,Masaki H,Machida M

    更新日期:2007-12-01 00:00:00

  • Bifunctional protein conferring enhanced green fluorescence and puromycin resistance.

    abstract::A new genetic marker was created in which sequences from enhanced green fluorescent protein were fused to those of puromycin N-acetyl transferase. The resulting fusion protein (EGFP-puro) conferred both green fluorescence and resistance to puromycin when expressed in mammalian cells. The utility of EGFP-puro as a sele...

    journal_title:BioTechniques

    pub_type:

    doi:10.2144/01312st05

    authors: Abbate J,Lacayo JC,Prichard M,Pari G,McVoy MA

    更新日期:2001-08-01 00:00:00

  • En masse analysis of nascent translation using microarrays.

    abstract::We report a robust method for studying en masse changes in translation using cDNA arrays. The relative distribution of messenger RNAs (mRNAs) along polysome gradients was monitored by performing cDNA array analysis of each gradient fraction and quantifying the mRNA translational status by regression analysis. Using th...

    journal_title:BioTechniques

    pub_type: 杂志文章

    doi:10.2144/05391ST01

    authors: Mazan-Mamczarz K,Kawai T,Martindale JL,Gorospe M

    更新日期:2005-07-01 00:00:00

  • pGATA: a positive selection vector based on the toxicity of the transcription factor GATA-1 to bacteria.

    abstract::The transcription factor GATA-1 is a zinc finger DNA-binding protein essential for the development of red blood cells. When we expressed different regions of the zinc finger domain in bacteria using an isopropyl-beta-D-thiogalactoside (IPTG) inducible system, growth of bacteria harboring the active DNA-binding domain ...

    journal_title:BioTechniques

    pub_type: 杂志文章

    doi:10.2144/19962004684

    authors: Trudel P,Provost S,Massie B,Chartrand P,Wall L

    更新日期:1996-04-01 00:00:00

  • High-quality RNA and DNA from flow cytometrically sorted human epithelial cells and tissues.

    abstract::Microarray technologies have made possible comprehensive analyses of nucleic acid sequence and expression. However, the technology to obtain efficiently high-quality RNA and DNA suitable for array analysis from purified populations of neoplastic cells from human tissues has not been well addressed. Microdissection can...

    journal_title:BioTechniques

    pub_type: 杂志文章

    doi:10.2144/02324rr06

    authors: Barrett MT,Glogovac J,Prevo LJ,Reid BJ,Porter P,Rabinovitch PS

    更新日期:2002-04-01 00:00:00