High-throughput Selection Research Articles

High-throughput selection of effective RNAi probes for gene silencing.

RNA interference (RNAi) is a process of sequence-specific posttranscriptional gene silencing mediated by double-stranded RNA. RNAi has recently emerged as a powerful genetic tool to analyze gene function in mammalian cells. The power of this method is limited however, by the uncertainty in predicting the efficacy of small interfering RNAs (siRNAs) in silencing a gene. This has imposed serious limitations not only for small-scale but also for high-throughput RNAi screening initiatives in mammalian systems. We have developed a reliable and quantitative approach for the rapid and efficient identification of the most effective siRNA against any gene. The efficacy of siRNA sequences is monitored by their ability to reduce the expression of cognate target-reporter fusions with easily quantified readouts. Finally, using micro array-based cell transfections, we demonstrate an unlimited potential of this approach in high-throughput screens for identifying effective siRNA probes for silencing genes in mammalian systems. This approach is likely to have implications in the use of RNAi as a reverse genetic tool for analyzing mammalian gene function on a genome-wide scale.

Automated High-Throughput mRNA Selection from Eukaryotic Total RNA

We describe a new technology (patent pending) for high-throughput selection of poly(A)+ RNA from total RNA. A novel binding solution is used to ensure the efficient and specific binding of mRNA to oligo(dT) magnetic beads with high stringency, virtually eliminating the non-specific binding of ribosomal RNA (rRNA) either to oligo(dT) beads or to the poly(A)+ RNA bound to the beads. As quantified by real-time RT-PCR, more than 99% of the rRNA is removed in a single round selection and mRNAs are fully recovered for both highly-expressed (GAPDH) and poorly-expressed (DDPK) genes from a few μg total RNA. The protocol is adaptable to any generic robotic workstation and takes ∼30 minutes to process 96 samples.

Automated High-Throughput mRNA Selection From Eukaryotic Total RNA

Automated High-Throughput mRNA Selection From Eukaryotic Total RNA

Parallel cloning, expression, purification and crystallization of human proteins for structural genomics.

54 human genes were selected as test targets for parallel cloning, expression, purification and crystallization. Proteins from these genes were selected to have a molecular weight of between 14 and 50 kDa, not to have a high percentage of hydrophobic residues (i.e. more likely to be soluble) and to have no known crystal structures and were not known to be subunits of heterocomplexes. Four proteins containing transmembrane regions were selected for comparative tests. To date, 44 expression clones have been constructed with the Gateway cloning system (Invitrogen, The Netherlands). Of these, 35 clones were expressed as recombinant proteins in Escherichia coli strain BL21 (DE3)-pLysS, of which 12 were soluble and four have been purified to homogeneity. Crystallization conditions were screened for the purified proteins in 96-well plates under oil. After further refinement with the same device or by the hanging-drop method, crystals were grown, with needle, plate and prism shapes. A 2.12 A data set was collected for protein NCC27. The results provide insights into the high-throughput target selection, cloning, expression and crystallization of human genomic proteins.

Phage display and colony filter screening for high-throughput selection of antibody libraries.

During the last 12 years, antibody combinatorial libraries have provided a new approach for the construction and production of reagents and drugs based on the human monoclonal antibodies. Studies employing antibodies or antibody mimics have become an important part of the explosive growth of proteomics. This places tremendous emphasis on the new approaches for faster library screening, improved methods of selection and evaluation of novel applications. The phage display system, together with its variants of ribosome and bacterial display, is the most extensively used method for the rapid screening of large antibody libraries. However, in the last two years the need to improve selection methods together with a complex patent situation regarding the phage display system, has also directed research towards the possibility of performing antibody selection by colony filter screening. Here, we summarise the results obtained by these different methods of selection comparing their efficacy and advantages.

High-Throughput Selection of Retrovirus Producer Cell Lines Leads to Markedly Improved Efficiency of Germ Line-Transmissible Insertions in Zebra Fish

High-Throughput Selection of Retrovirus Producer Cell Lines Leads to Markedly Improved Efficiency of Germ Line-Transmissible Insertions in Zebra Fish

Synthesis, properties, and reactivity of cocaine benzoylthio ester possessing the cocaine absolute configuration.

One aspect of immunopharmacotherapy for cocaine abuse involves the use of a catalytic monoclonal antibody (mAb) to degrade cocaine via hydrolysis of the benzoate ester. A cocaine benzoylthio ester analogue provides a means to implement high-throughput selection strategies to potentially isolate mAbs with high activity. The required analogue was synthesized starting from (-)-cocaine hydrochloride and possessed the cocaine absolute configuration. Key points in the preparation were the introduction of the sulfur atom at C-3 via a bromomagnesium thiolate addition to the exo face of anhydroecgonine, separation of C-2 diastereomers, recycling of a C-2 thio ester byproduct, and formation of the necessary C-2 methyl and C-3 benzoylthio esters. Effects resulting from the lower electronegativity and greater hydrophobicity of sulfur compared to oxygen were observed. These characteristics could result in interesting drug properties. Furthermore, the analogue was found to be a substrate for catalytic mAbs that hydrolyze cocaine as monitored by HPLC and also spectrophotometry by coupling cleavage of the benzoylthio ester to the disulfide exchange with Ellman's reagent. Screening antibody libraries with the new cocaine analogue using the spectroscopic assay provides an avenue for the high-throughput identification of catalysts that efficiently breakdown cocaine.

Mutagenic polymerase chain reaction of protein-coding genes for in vitro evolution.

In vitro protein evolution is an efficient approach to study the structure and function of a protein, or to enhance its industrial utility (). One round of evolution consists of random mutation of a protein-coding gene, expression the resulting library in a population of micro-organisms, and high-throughput screening or selection of clones that most strongly exhibit a desired phenotype (“winners”). After many rounds, mutations that confer the phenotype accumulate on a single allele, e.g., the authors have isolated an octuple mutant of the Escherichia coli β-glucuronidase with catalytic activity resistant to roughly 80-fold higher concentrations of glutaraldehyde than that of the wild-type enzyme (). Here we describe a variation of the mutagenic polymerase chain reaction (PCR) (,) is described. The advantages of this method over other random mutagenesis techniques are explained.

Antibodies in haystacks: how selection strategy influences the outcome of selection from molecular diversity libraries

Antibodies against most antigens can be isolated from high quality phage antibody libraries. However, not all antibodies binding a particular antigen are necessarily found when standard selections are performed. Here we investigate the effect of two different selection strategies on the isolation of antibodies against a number of different antigens, and find that these different strategies tend to select different antibodies, with little overlap between them. This indicates that the full diversity of these libraries is not tapped by a single selection strategy and that each selection strategy imposes different selective criteria in addition to that of antigen binding. To fully exploit such libraries, therefore, many different selection strategies should probably be employed for each antigen. The use of alternative strategies should be considered when selection apparently fails, or when the number of different antibodies recognizing an antigen needs to be maximised. Furthermore, the microtitre selection strategy developed is likely to prove useful in the application of phage antibody libraries to the human genome project, allowing the high throughput selection of antibodies against multiple antigens simultaneously.

Synthetic promoter elements obtained by nucleotide sequence variation and selection for activity

Eukaryotic transcriptional regulation in different cells involves large numbers and arrangements of cis and trans elements. To survey the number of cis regulatory elements that are active in different contexts, we have devised a high-throughput selection procedure permitting synthesis of active cis motifs that enhance the activity of a minimal promoter. This synthetic promoter construction method (SPCM) was used to identify >100 DNA sequences that showed increased promoter activity in the neuroblastoma cell line Neuro2A. After determining DNA sequences of selected synthetic promoters, database searches for known elements revealed a predominance of eight motifs: AP2, CEBP, GRE, Ebox, ETS, CREB, AP1, and SP1/MAZ. The most active of the selected synthetic promoters contain composites of a number of these motifs. Assays of DNA binding and promoter activity of three exemplary motifs (ETS, CREB, and SP1/MAZ) were used to prove the effectiveness of SPCM in uncovering active sequences. Up to 10% of 133 selected active sequences had no match in currently available databases, raising the possibility that new motifs and transcriptional regulatory proteins to which they bind may be revealed by SPCM. The method may find uses in constructing databases of active cis motifs, in diagnostics, and in gene therapy.

Synthetic promoter elements obtained by nucleotide sequence variation and selection for activity

Eukaryotic transcriptional regulation in different cells involves large numbers and arrangements of cis and trans elements. To survey the number of cis regulatory elements that are active in different contexts, we have devised a high-throughput selection procedure permitting synthesis of active cis motifs that enhance the activity of a minimal promoter. This synthetic promoter construction method (SPCM) was used to identify >100 DNA sequences that showed increased promoter activity in the neuroblastoma cell line Neuro2A. After determining DNA sequences of selected synthetic promoters, database searches for known elements revealed a predominance of eight motifs: AP2, CEBP, GRE, Ebox, ETS, CREB, AP1, and SP1/MAZ. The most active of the selected synthetic promoters contain composites of a number of these motifs. Assays of DNA binding and promoter activity of three exemplary motifs (ETS, CREB, and SP1/MAZ) were used to prove the effectiveness of SPCM in uncovering active sequences. Up to 10% of 133 selected active sequences had no match in currently available databases, raising the possibility that new motifs and transcriptional regulatory proteins to which they bind may be revealed by SPCM. The method may find uses in constructing databases of active cis motifs, in diagnostics, and in gene therapy.

Biopanning phage display libraries using magnetic beads vs. polystyrene plates.

BioTechniquesVol. 26, No. 2 BenchmarksOpen AccessBiopanning Phage Display Libraries Using Magnetic Beads Vs. Polystyrene PlatesStephen J. McConnell, Thai Dinh, Mong-Huong Le & Dominic G. SpinellaStephen J. McConnell*Address correspondence to Dr. Stephen J. McConnell, Chugai Biopharmaceuticals, 6275 Nancy Ridge Drive, San Diego, CA 92121, USA. Internet: E-mail Address: smcconnell@chugaibio.comChugai Biopharmaceuticals, San Diego, CA, USASearch for more papers by this author, Thai DinhChugai Biopharmaceuticals, San Diego, CA, USASearch for more papers by this author, Mong-Huong LeChugai Biopharmaceuticals, San Diego, CA, USASearch for more papers by this author & Dominic G. SpinellaChugai Biopharmaceuticals, San Diego, CA, USASearch for more papers by this authorPublished Online:22 Aug 2018https://doi.org/10.2144/99262bm06AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail FiguresReferencesRelatedDetailsCited ByGigavalent Display of Proteins on Monodisperse Polyacrylamide Hydrogels as a Versatile Modular Platform for Functional Assays and Protein Engineering1 August 2022 | ACS Central Science, Vol. 8, No. 8Evaluation of Phage Display Biopanning Strategies for the Selection of Anti-Cell Surface Receptor Antibodies30 July 2022 | International Journal of Molecular Sciences, Vol. 23, No. 15Emerging ELISA derived technologies for in vitro diagnosticsTrAC Trends in Analytical Chemistry, Vol. 152Auto-Panning: a highly integrated and automated biopanning platform for peptide screening1 January 2021 | Lab on a Chip, Vol. 21, No. 14Development of phage biopanning strategies to identify affinity peptide ligands for kappa light chain Fab fragments25 July 2019 | Biotechnology Progress, Vol. 35, No. 6Phage display antibody libraries: A robust approach for generation of recombinant human monoclonal antibodiesInternational Journal of Biological Macromolecules, Vol. 135Invert biopanning: A novel method for efficient and rapid isolation of scFvs by phage display technologyBiologicals, Vol. 44, No. 6Continuous microfluidic assortment of interactive ligands (CMAIL)31 August 2016 | Scientific Reports, Vol. 6, No. 1Calf thymus histone-conjugated magnetic poly(2-oxoethyl methacrylate) microspheres for affinity isolation of anti-histone IgGs from the blood serum of patients with systemic lupus erythematosus1 January 2015 | RSC Advances, Vol. 5, No. 77Affinity Selection of Peptide Binders with Magnetic Beads via Organic Phase Separation (MOPS)Biological & Pharmaceutical Bulletin, Vol. 38, No. 11Micro/Nano Technologies and Their Biological and Medical ApplicationsPhage ESCape: An emulsion-based approach for the selection of recombinant phage display antibodiesJournal of Immunological Methods, Vol. 367, No. 1-2Generation of Fab Fragment-Like Molecular Recognition Proteins against Staphylococcal Enterotoxin B by Phage Display TechnologyClinical and Vaccine Immunology, Vol. 17, No. 11Generation of a Human Anti-Tumor Necrosis Factor-α Monoclonal Antibody by in Vitro Immunization with a Multiple Antigen Peptide22 May 2014 | Bioscience, Biotechnology, and Biochemistry, Vol. 74, No. 9Automation in the High-throughput Selection of Random Combinatorial Libraries—Different Approaches for Select Applications8 April 2010 | Molecules, Vol. 15, No. 4A Novel Strategy for Proteome-wide Ligand Screening Using Cross-linked Phage MatricesJournal of Biological Chemistry, Vol. 285, No. 13Production of Human Monoclonal Antibodies against FcεRIα by a Method Combining in Vitro Immunization with Phage Display22 May 2014 | Bioscience, Biotechnology, and Biochemistry, Vol. 73, No. 7Automated Panning and Screening Procedure on Microplates for Antibody Generation from Phage Display LibrariesSLAS Discovery, Vol. 14, No. 3A rapid and efficient strategy to generate antigen-specific human monoclonal antibody by in vitro immunization and the phage display methodJournal of Immunological Methods, Vol. 332, No. 1-2Magnetic moment measurement of magnetic nanoparticles using atomic force microscopy17 December 2007 | Measurement Science and Technology, Vol. 19, No. 1Addressing the Protein Crystallization Bottleneck By Cocrystallization23 October 2007 | Crystal Growth & Design, Vol. 7, No. 11Templated synthesis of gold–iron alloy nanoparticles using pulsed laser deposition28 September 2006 | Nanotechnology, Vol. 17, No. 20Synthesis and Characterization of Paramagnetic Microparticles through Emulsion-Templated Free Radical Polymerization16 February 2006 | Langmuir, Vol. 22, No. 6Phage-display selection of antibodies to the left end of CTX3C using synthetic fragmentsJournal of Immunological Methods, Vol. 289, No. 1-2Phage display for epitope determination: A paradigm for identifying receptor–ligand interactionsMagnetic-bead enzyme-linked immunosorbent assay verifies adsorption of ligand and epitope accessibilityAnalytical Biochemistry, Vol. 311, No. 2Automation of phage display for high-throughput antibody developmentTARGETS, Vol. 1, No. 1Phage display combinatorial libraries of short peptides: ligand selection for protein purificationEnzyme and Microbial Technology, Vol. 28, No. 9-10Bead-based HTS applications in drug discoveryDrug Discovery Today, Vol. 5 Vol. 26, No. 2 Follow us on social media for the latest updates Metrics Downloaded 1,416 times History Published online 22 August 2018 Published in print February 1999 Information© 2018 Author(s)PDF download