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BioTechniques

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https://www.readbyqxmd.com/read/28403810/transient-dominant-host-range-selection-using-chinese-hamster-ovary-cells-to-generate-marker-free-recombinant-viral-vectors-from-vaccinia-virus
#1
Liang Liu, Tamara Cooper, Preethi Eldi, Pablo Garcia-Valtanen, Kerrilyn R Diener, Paul M Howley, John D Hayball
Recombinant vaccinia viruses (rVACVs) are promising antigen-delivery systems for vaccine development that are also useful as research tools. Two common methods for selection during construction of rVACV clones are (i) co-insertion of drug resistance or reporter protein genes, which requires the use of additional selection drugs or detection methods, and (ii) dominant host-range selection. The latter uses VACV variants rendered replication-incompetent in host cell lines by the deletion of host-range genes. Replicative ability is restored by co-insertion of the host-range genes, providing for dominant selection of the recombinant viruses...
April 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28403809/effective-dna-fragmentation-technique-for-simple-sequence-repeat-detection-with-a-microsatellite-enriched-library-and-high-throughput-sequencing
#2
Keisuke Tanaka, Rumi Ohtake, Saki Yoshida, Takashi Shinohara
Two different techniques for genomic DNA fragmentation before microsatellite-enriched library construction-restriction enzyme (NlaIII and MseI) digestion and sonication-were compared to examine their effects on simple sequence repeat (SSR) detection using high-throughput sequencing. Tens of thousands of SSR regions from 5 species of the plant family Myrtaceae were detected when the output of individual samples was >1 million paired-end reads. Comparison of the two DNA fragmentation techniques showed that restriction enzyme digestion was superior to sonication for identification of heterozygous genotypes, whereas sonication was superior for detection of various SSR flanking regions with both species-specific and common characteristics...
April 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28403808/imagej-macros-for-the-user-friendly-analysis-of-soft-agar-and-wound-healing-assays
#3
João Paulo Silva Nunes, Adriana Abalen Martins Dias
Recent advances in biological imaging techniques and the enormous amount of data they generate call for the development of computational tools for efficient and reliable high-throughput analysis. Several software applications with this functionality are available, and one of the most commonly used is ImageJ. Here, we present two independent macros (WH_NJ and SA_NJ) for automating and facilitating the analysis of images acquired from two in vitro assays frequently used in cancer studies and drug screening: the wound-healing and soft-agar assays...
April 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28403807/generating-stable-cell-lines-with-quantifiable-protein-production-using-crispr-cas9-mediated-knock-in
#4
Chiu-An Lo, Alexander W Greben, Brian Edwin Chen
Cell lines expressing foreign genes have been widely used to produce a variety of recombinant proteins. However, generating recombinant protein-expressing cell lines is usually a lengthy process and the resulting protein expression levels are often inconsistent. Here, we describe an efficient method for making stable cell lines expressing any recombinant protein of interest in a controllable and quantifiable manner. We integrate transgenes into specific genomic loci using CRISPR/Cas9 such that transgene expression is driven by endogenous promoters to ensure consistent and predictable expression of the recombinant protein...
April 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28403806/two-color-fluorescent-cytosine-extension-assay-for-the-determination-of-global-dna-methylation
#5
Gu Zhou, Craig Parfett, Cathy Cummings-Lorbetskie, Gong-Hua Xiao, Daniel Desaulniers
Here, we present a DNA restriction enzyme-based, fluorescent cytosine extension assay (CEA) to improve normalization and technical variation among sample-to-sample measurements. The assay includes end-labeling of parallel methylation-sensitive and methylation-insensitive DNA restriction enzyme digests along with co-purification and subsequent co-measurement of incorporated fluorescence. This non-radioactive, two-color fluorescent CEA (TCF-CEA) was shown to be a relatively rapid and accurate, with 3-fold greater precision than the one-color CEA...
April 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28403805/entering-the-matrix
#6
Amber Dance
Animal cells interact with the extracellular matrix to divide, manage shape, and migrate. Amber Dance looks at new techniques aimed at revealing how this happens.
April 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28403804/pcr-s-next-wave
#7
Nathan Blow
New applications are driving the need for faster, higher fidelity PCR. Nathan Blow looks at some clever modifications taking PCR to the next level.
April 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28298179/preparation-of-peptide-mhc-and-t-cell-receptor-dextramers-by-biotinylated-dextran-doping
#8
Michael T Bethune, Begoña Comin-Anduix, Yu-Hsien Hwang Fu, Antoni Ribas, David Baltimore
Peptide-major histocompatibility complex (pMHC) multimers enable the detection, characterization, and isolation of antigen-specific T-cell subsets at the single-cell level via flow cytometry and fluorescence microscopy. These labeling reagents exploit a multivalent scaffold to increase the avidity of individually weak T-cell receptor (TCR)-pMHC interactions. Dextramers are an improvement over the original streptavidin-based tetramer technology because they are more multivalent, improving sensitivity for rare, low-avidity T cells, including self/tumor-reactive clones...
March 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28298178/antibody-incubation-at-37%C3%A2-c-improves-fluorescent-immunolabeling-in-free-floating-thick-tissue-sections
#9
Xia Xiao, Ya-Ping Feng, Bin Du, Han-Ru Sun, You-Quan Ding, Jian-Guo Qi
Fluorescent immunolabeling and imaging in free-floating thick (50-60 μm) tissue sections is relatively simple in practice and enables design-based non-biased stereology, or 3-D reconstruction and analysis. This method is widely used for 3-D in situ quantitative biology in many areas of biological research. However, the labeling quality and efficiency of standard protocols for fluorescent immunolabeling of these tissue sections are not always satisfactory. Here, we systematically evaluate the effects of raising the conventional antibody incubation temperatures (4°C or 21°C) to mammalian body temperature (37°C) in these protocols...
March 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28298177/tympanic-membrane-organ-culture-using-cell-culture-well-inserts-engrafted-with-tympanic-membrane-tissue-explants
#10
Lawrence J Liew, Richard M Day, Rodney J Dilley
Tissue engineering approaches using growth factors and various materials for repairing chronic perforations of the tympanic membrane are being developed, but there are surprisingly few relevant tissue culture models available to test new treatments. Here, we present a simple three-dimensional model system based on micro-dissecting the rat tympanic membrane umbo and grafting it into the membrane of a cell culture well insert. Cell outgrowth from the graft produced sufficient cells to populate a membrane of similar surface area to the human tympanic membrane within 2 weeks...
March 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28298176/semi-automated-tip-snip-cloning-of-restriction-fragments-into-and-out-of-plasmid-polylinkers
#11
Ichiro Matsumura
Synthetic biologists rely on semi-synthetic recombinant plasmids, but DNA synthesis is constrained by practical limits on length, accuracy, and sequence composition. Cloned DNA parts can be assembled into longer constructs via subcloning, but conventional methods are labor-intensive. One-pot recombination reactions are more convenient but harder to troubleshoot, and those that depend on PCR to create fragments with compatible ends necessitate re-sequencing. The Tip Snip protocol described here enables the subcloning of an insert from one plasmid polylinker into another without PCR or gel purification steps...
March 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28298175/cells-in-the-third-dimension
#12
Sarah Webb
Sarah Webb explores the expanding tools and technologies for 3-D cell culture-from hydrogels to organs-on-a-chip.
March 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28193152/a-fast-method-for-analyzing-essential-protein-mutants-in-human-cells
#13
Frank Dietsch, Mariel Donzeau, Agnes M Cordonnier, Etienne Weiss, Bruno Chatton, Marc Vigneron
Here we developed a complementation method for the study of essential genes in live human cells using the CRISPR/Cas9 system. Proteins encoded by essential genes were expressed using a derivative of the pCEP4 compensating plasmid in combination with Cas9 endonuclease targeting of the chromosomal genes. We show that this strategy can be applied to essential genes, such as those coding for proliferating cell nuclear antigen (PCNA) and DNA polymerase delta subunit 2 (POLD2). As demonstrated for the PCNA protein, our method allows mutational analysis of essential protein-coding sequences in live cells...
February 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28193151/extraction-of-ultrashort-dna-molecules-from-herbarium-specimens
#14
Rafal M Gutaker, Ella Reiter, Anja Furtwängler, Verena J Schuenemann, Hernán A Burbano
DNA extracted from herbarium specimens is highly fragmented; therefore, it is crucial to use extraction protocols that retrieve short DNA molecules. Improvements in extraction and DNA library preparation protocols for animal remains have allowed efficient retrieval of molecules shorter than 50 bp. Here, we applied these improvements to DNA extraction protocols for herbarium specimens and evaluated extraction performance by shotgun sequencing, which allows an accurate estimation of the distribution of DNA fragment lengths...
February 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28193150/maleimide-scavenging-enhances-determination-of-protein-s-palmitoylation-state-in-acyl-exchange-methods
#15
Charlotte H Hurst, Dionne Turnbull, Fiona Plain, William Fuller, Piers A Hemsley
S-palmitoylation (S-acylation) is emerging as an important dynamic post-translational modification of cysteine residues within proteins. Current assays for protein S-palmitoylation involve either in vivo labeling or chemical cleavage of S-palmitoyl groups to reveal a free cysteine sulfhydryl that can be subsequently labeled with an affinity handle (acyl-exchange). Assays for protein S-palmitoylation using acyl-exchange chemistry therefore require blocking of non-S-palmitoylated cysteines, typically using N-ethylmaleimide (NEM), to prevent non-specific detection...
February 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28193149/taqman-probes-as-blocking-agents-for-enriched-pcr-amplification-and-dna-melting-analysis-of-mutant-genes
#16
Irina V Botezatu, Irina O Panchuk, Anna M Stroganova, Anastasia I Senderovich, Valentina N Kondratova, Valery P Shelepov, Anatoly V Lichtenstein
Asymmetric PCR and DNA melting analysis with TaqMan probes applied for mutation detection is effectively used in clinical diagnostics. The method is simple, cost-effective, and carried out in a closed-tube format, minimizing time, labor, and risk of sample cross-contamination. Although DNA melting analysis is more sensitive than Sanger sequencing (mutation detection thresholds are ~5% and 15%-20%, respectively), it is less sensitive than more labor-intensive and expensive techniques such as pyrosequencing and droplet digital PCR...
February 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28193148/external-calibration-with-drosophila-whole-cell-spike-ins-delivers-absolute-mrna-fold-changes-from-human-rna-seq-and-qpcr-data
#17
Franziska Taruttis, Maren Feist, Phillip Schwarzfischer, Wolfram Gronwald, Dieter Kube, Rainer Spang, Julia C Engelmann
Gene expression measurements are typically performed on a fixed-weight aliquot of RNA, which assumes that the total number of transcripts per cell stays nearly constant across all conditions. In cases where this assumption does not hold (e.g., when comparing cell types with different cell sizes) the expression data provide a distorted view of cellular events. Assuming constant numbers of total transcripts, increases in expression of some RNAs must be compensated for by decreases in expression of others. Therefore, we propose calibrating gene expression data to an external reference point, the number of cells in the sample, using whole-cell spike-ins...
February 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28193147/print-on-demand
#18
Sarah Webb
Advances in 3-D printing and biomaterials are helping to shape the field of regenerative medicine. Sarah Webb examines the latest developments.
February 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28118814/bxb1-phage-recombinase-assists-genome-engineering-in-drosophila-melanogaster
#19
Roumen Voutev, Richard S Mann
Rapid and reliable genome modifications provide the basis for detailed in vivo functional analysis of any genomic entity (gene, regulatory DNA, non-coding RNA, etc). With the advent of CRISPR/Cas9 genome editing technology, manipulation of a particular genomic locus has become a routine undertaking in variety of model organisms, including the fruit fly Drosophila melanogaster. To further diversify the available tools for genome engineering, we successfully harnessed the phage recombinase Bxb1 to perform recombinase-mediated cassette exchange (RMCE) in D...
January 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28118813/tumblescore-run-and-tumble-analysis-for-low-frame-rate-motility-videos
#20
Alex Eli Pottash, Ryan McKay, Chelsea R Virgile, Hana Ueda, William E Bentley
Scientists often exploit the motility of peritrichously flagellated bacteria for various applications. A common alteration is modifying the frequency of mid-movement changes in direction, known as tumbles. Such differences in bacterial swimming patterns can prove difficult to quantify, especially for those without access to high-speed optical equipment. Traditionally, scientists have resorted to less accurate techniques, such as soft agar plate assays, or have been forced to invest in costly equipment. Here, we present TumbleScore, software designed to track and quantify bacterial movies with slow, as well as fast, frame-rates...
January 1, 2017: BioTechniques
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