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BioTechniques

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https://www.readbyqxmd.com/read/28528577/single-step-pcr-based-genetic-sex-determination-of-rat-tissues-and-cells
#1
Pramod Dhakal, Michael J Soares
The advent of genome editing strategies has expanded the range of animal models available for gene manipulation and renewed research interest in the rat. Gender is a key variable for in vivo gene function analyses. Here, we present a simple PCR-based method to determine genetic sex in the rat.
May 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28528576/using-non-thermal-irreversible-electroporation-to-create-an-in-vivo-niche-for-exogenous-cell-engraftment
#2
Tammy T Chang, Vivian X Zhou, Boris Rubinsky
The critical shortage of donor organs has spurred investigation of alternative approaches to either generate replacement organs or implant exogenous cells for treatment of end-stage organ failure. Non-thermal irreversible electroporation (NTIRE), which uses brief high electric field pulses to induce irreversible permeabilization of cell membranes, has emerged as a technique for tumor ablation. Here, we introduce a new application for NTIRE that employs in situ cell ablation to create a niche within a solid organ for engraftment of exogenous cells in vivo...
May 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28528575/characterization-of-a-cyclic-olefin-polymer-microcentrifuge-tube
#3
Lloyd Waxman, Rachel L Erwin, Vinod D Vilivalam
Here, we describe the properties of a prototype microcentrifuge tube made from the plastic cyclic olefin polymer (COP). This material has been used in the manufacture of primary containers including syringes and vials for the storage, shipment, and delivery of biotherapeutics, vaccines, and cell therapy products. Its low level of extractable substances and metals along with its glass-like clarity make COP an attractive material for the fabrication of microcentrifuge tubes and other consumable laboratory plasticware where contamination is an important consideration, such as in the storage and analysis of labile proteins, nucleic acids, and metabolites...
May 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28528574/esc-track-a-computer-workflow-for-4-d-segmentation-tracking-lineage-tracing-and-dynamic-context-analysis-of-escs
#4
Laura Fernández-de-Manúel, Covadonga Díaz-Díaz, Daniel Jiménez-Carretero, Miguel Torres, María C Montoya
Embryonic stem cells (ESCs) can be established as permanent cell lines, and their potential to differentiate into adult tissues has led to widespread use for studying the mechanisms and dynamics of stem cell differentiation and exploring strategies for tissue repair. Imaging live ESCs during development is now feasible due to advances in optical imaging and engineering of genetically encoded fluorescent reporters; however, a major limitation is the low spatio-temporal resolution of long-term 3-D imaging required for generational and neighboring reconstructions...
May 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28528573/a-sensitive-semi-quantitative-mammalian-two-hybrid-assay
#5
Elisabeth Riegel, Thomas Heimbucher, Thomas Höfer, Thomas Czerny
Protein-protein interactions critically determine the function of a protein within the cell. Several methods have been developed for the analysis of protein interactions, including two-hybrid assays in yeast and mammals. Mammalian two-hybrid systems provide the ideal physiological environment to study the interactions of mammalian proteins; however, these approaches are limited in sensitivity and their ability to quantify interaction strength. Here, we present an inducible mammalian two-hybrid (iM2H) system using the small-molecule dimerizer rapalog for recruitment of multiple transactivation domains into the M2H system...
May 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28528572/going-with-the-flow
#6
Nathan Blow
From bacteria to circulating tumor cells, advances in flow cytometry technology are pushing the boundaries of cell biology research.
May 1, 2017: BioTechniques
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
#7
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
#8
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
#9
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
#10
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
#11
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
#12
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
#13
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
#14
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
#15
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
#16
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
#17
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
#18
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
#19
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
#20
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
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