BioSpotlight
FADE RESISTANT CULTURING When it comes to fluorescent proteins (FP) and their usage in cell biology, photostability is a key factor. If fluorescence decreases rapidly or is low to begin with, obtaining biologically relevant data from an experiment can be difficult. In an effort to better understand photostability issues associated with cells in culture, Konstantin Lukyanov and colleagues from the Institute of Bioorganic Chemistry in Moscow, Russia systematically evaluated the influence of growth conditions and media composition on green fluorescent protein (GFP) stability. The authors examined the components in Ham’s F12 culture medium that might enhance GFP photostability, finding that decreased concentrations of riboflavin and pyridoxine but increased concentrations of other components compared to DMEM were key to generating greater FP photostability. They also showed that cell density and serum concentration have profound effects on photostability. Taken together, Lukyanov and his co-authors provide a set of guidelines for researchers looking to optimize FP photostability for their cell culture experiments and ultimately get the most meaningful data out of FP experiments.
See “Influence of cell growth conditions and medium compo- sition on EGFP photostability in live cells” on page 258.
FINDING THAT BETTER MATE Although the number of sequenced genomes is quickly rising, there are still many species for which a reference genome remains missing. When it comes to de novo genome sequencing in these cases, mate-pair reads are essential to obtaining the necessary scaffolding data that will ultimately result in a genome assembly, but generating good mate-pair libraries for this purpose remains a challenging task. In this issue of BioTechniques, a team of researchers from the RIKEN Center for Developmental Biology, led by Shigehiro Kuraku, describe a series of modifications for one of the most popular mate-pair library kits, the Nextera Mate Pair Sample Prep Kit, that should enhance both yield and efficiency. Through intensive DNA shearing to decrease insert sizes, sequencing with >150 cycles, and the use of variable tagmentation conditions, Kuraku and his colleagues were able to demonstrate clear increases in overall sequence continuity while at the same time reducing the costs associated with generating mate-pair libraries.
See “Optimization and cost-saving in tagmentation-based mate-pair library preparation and sequencing” on page 253.
Selected and written by Nathan S. Blow, Ph.D. BioTechniques 58:215 (May 2015) doi 10.2144/000114282
Vol. 58 | No. 5 | 2015 215
Citations
VISUALIZING PROTEOME-LEVEL METABOLIC DYNAMICS Most methods for monitoring global protein metabolism in cell culture lack spatial information or require cell fixation. Previously, Wei et al. described a live imaging technique based on stimu- lated Raman scattering (SRS) detection of the carbon-deuterium bond in cells metabolically labeled with deuterated amino acids. While promising, the method was relatively insensitive and could only assay protein synthesis. Now the authors have improved the sensitivity and, by examining the vibrational signature of methyl groups in pre-existing protein pools, their approach can also monitor protein degradation. Even pulse-chase experiments are possible, by sequentially exposing cells to non-branched and then branched deuterated amino acids (the two subtypes can be distin- guished by SRS). The new method works in cell culture, live brain tissue slices, zebrafish embryos, and mice. It should be especially well-suited for exploring protein synthesis related to memory formation and protein metabolism in animal models of disease.
L. Wei et al. 2015. Imaging complex protein metabolism in live organisms by stimulated Raman scattering microscopy with isotope labeling. ACS Chem Biol. 10(3):901-8.
TISSUE-SPECIFIC GENOME EDITING IN ZEBRAFISH Loss-of-function studies in zebrafish have come a long way from random mutagenesis screens. However, CRISPR/Cas9-based genome editing results in global inactivation of the target gene. Ablain et al. now report a vector system for tissue-specific gene inactivation in zebrafish. In their integratable vector, which is microinjected into one-cell-stage embryos, the guide RNA is ubiquitously expressed, while Cas9 is driven by a tissue-specific promoter. Tests with an erythrocyte-specific promoter showed blood-lineage knockout of an enzyme involved in heme biosyn- thesis in both F0 embryos and F1 offspring selected for presence of the integrated vector. Moreover, experiments involving a vector targeting p53 confirmed the system is also applicable to genetic suppressor screens. The authors anticipate that their vector will be compatible with targeting multiple genes (via incorporation of several guide RNA units) and that the overall approach may be trans- ferable to other vertebrate models such as Xenopus and medaka.
J. Albain et al. 2015. A CRISPR/Cas9 vector system for tissue- specific gene disruption in zebrafish. Dev Cell. 32(6):756-64.
Selected and written by Nijsje Dorman, Ph.D. BioTechniques 58:215 (May 2015) doi 10.2144/000114282
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