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Summary of the January 2016 Issue of BioTechniques

The January 2016 issue of BioTechniques will feature new methods and techniques, including:

  • a new tool for the generation of transgenic zebrafish
  • a simple to implement device for applying small forces to biomolecules
  • a new strategy for assaying viruses using PCR
  • a novel technique for obtaining breast milk for research studies without the need for oxytocin injections
  • a unique high-throughput platform to study tumor spheroids
  • an optical tweezers-based methodology for the temporal and spatial analysis of extracellular vesicles

In addition to these new methods articles, January will also contain a new Tech News feature focusing on the latest in high-content screening technologies and instrumentation.

 Article Descriptions

Transgenic zebrafish have enhanced researchers abilities to perform large-scale genetic and small molecule screens for gene and drug discovery. In the January 2016 issue of BioTechniques, researchers from Boston University in Boston, MA report a new, efficient method for generating transgenic zebrafish that utilizes pigmentation rescue of nacre mutant fish for accurate naked-eye identification of both mosaic founders and stable transgenic zebrafish. The authors found that pigmentation rescue reliably predicts transgene integration, creating a reliable means to screen for transgenic zebrafish and enhancing genetic screen capabilities.

Keywords: zebrafish; genetic screening; small molecule screens; transgenic animals

The manipulation of DNA and proteins using biophysical methods has led to unique insights into single molecule biology. In January, a team from Michigan reports the development of a simple-to-implement magnetic force transducer that can apply a wide range of piconewton scale forces on single DNA molecules and DNA-protein complexes in the horizontal plane. The resulting low-noise force-extension data enables very high-resolution detection of changes in the DNA tether’s extension: ~ 0.05 pN in force and less than 10 nm changes in extension, allowing for the detailed dissection of the physical properties of biomolecules.

Keywords: atomic force microscopy; cell motility; DNA extension; DNA-protein complexes

High sequence variation in RNA viruses necessitates use of degenerate primers and probes or multiple primers and probes for molecular diagnostic assays. In this January article, a research team from Sweden demonstrates that long primers and probes of up to 56 nucleotides can also be applied in real-time PCR for detection of norovirus genogroup II, improving assay sensitivity.

Keywords: real-time PCR; virus detection; molecular diagnostics; viral analysis

It has been reported that breastfeeding over 6 months strongly decreases the risk of allergy, diabetes, obesity and hypertension. In order to understand the mechanisms responsible for this benefit, it is important to precisely evaluate the composition of maternal milk, especially following environmental cues. Taking advantage of a natural stimulation of mammary gland in mouse pups, researchers describe their development of a new milking procedure that collects mouse milk without the need for oxytocin injection to stimulate production. The method is easy to use, low-cost and non-invasive, providing sufficient material to allow a large range of biological analyses.

Keywords: animal models; oxytocin injection; mouse models

Tumor spheroids are becoming an important tool for the investigation of cancer stem cell function in tumors, but low-cost high throughput methods for tumor spheroids screening are still needed. Using non-adherent three-dimensional (3D) cultures, researchers from Northwestern University in Chicago developed a simple acridine orange-based method that allows for rapid analysis of live tumor spheroids by fluorescence microscopy in a 96-well format. The method is suitable for monitoring the effect of antineoplastic drugs on neurosphere formation as both rapamycin and erlotinib attenuated neurosphere size thus allowing for rapid screening of anti-proliferative drugs to assess their ability in inhibiting growth of cancer stem cells as 3D structures.

Keywords: cancer research; tumor spheroids; 3D cell cultures; fluorescence microscopy; image analysis; drug screening

Extracellular vesicles are circular membrane structures released by most cells that represent highly conserved mediators of intercellular communication. However, temporal and spatial dynamics of vesicle-cell interaction still remain largely unexplored. In a Report in the January issue, researchers from Italy used optical tweezers to drive single extracellular vesicles produced by microglial cells on the surface of astrocytes or microglia in primary culture. By visualizing single extracellular vesicle-cell contacts, microglial vesicles were found to display different motilities on the surface of astrocytes or microglia. The data identify optical manipulation as a powerful tool to monitor in vitro vesicle-cell dynamics with temporal and spatial resolution, and determine in a quantitative manner the contribution of surface receptors /extracellular protein ligands to the contact.

Keywords: extracellular vesicles; cell-cell communication; optical tweezers; biophysical analysis; microscopy; image analysis

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