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BioTechniquesVol. 56, No. 3 BioSpotlightOpen AccessBioSpotlightNathan BlowNathan BlowSearch for more papers by this authorPublished Online:3 Apr 2018https://doi.org/10.2144/000114140AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail Bridging the gap in chromatin analysisDetermining the structure and organization of chromatin within cells is important for understanding how gene expression is regulated and how genomes evolve. While a number of techniques, including fluorescence in situ hybridization (FISH) and various chromatin conformation capture analyses (e.g., 3C and Hi-C), have been developed to study chromatin organization and interactions, each of these approaches has drawbacks. While FISH allows single-cell studies of chromatin interactions, the resolution of this technique can be limited to the diffraction limit of light, ∼250 nm. Super resolution microscopy techniques have improved upon this limitation, but still not to sufficient resolution to discern specific chromatin features or enable three-dimensional reconstruction of chromatin interactions. Chromatin conformation capture techniques enable much higher resolution studies of chromatin interactions but also require large numbers of cells, resulting in an inability to assess the frequencies of specific interactions within individual cells. In an effort to bridge the gap between the high resolution of chromatin conformation analysis and the single cell perspective provided by FISH, Chen et al. (Karolinska Institute, Stockholm, Sweden) present a new technique, chromatin in situ proximity (ChrISP), in the current issue of BioTechniques. The methodological basis of ChrISP lies in the well-developed in situ proximity ligation assay (ISPLA), wherein two probes create a fluorescent amplification signal when in close proximity to one another. Two forms of ChrISP were described by the authors: In one, rolling circle amplification (RCA) is used to amplify the signal between the two probes, while the second avoids RCA, instead relying on labeled “splinter” and “backbone” oligos to create a signal between the two chromatin probes. The authors demonstrate that by using the “Green-Splinter ChrISP” (splinter plus backbone oligos with no RCA) they were able to obtain resolutions greater than 17 nm in all 3 dimensions, a 30-fold improvement in the Z-plane in comparison to the other approaches. In the end, given the versatility and simplicity of the ChrISP technique, Chen et al. predict that the method will prove extremely useful when examining chromatin structure alone as well as the alignment of chromatin with other cell landmarks, such as the nuclear membrane and nucleoli.See “Chromatin in situ proximity (ChrISP): Single-cell analysis of chromatin proximities at high resolution”Needle-ing butterfliesAfter years of development and optimization, it is easy for Drosophila researchers to take germline transformation for granted. While it is a demanding technique that quite often requires a large number of injections for success, a transformant can usually be obtained. For other species though, the tools for germline transformation are less well developed, presenting a challenge for scientists wanting to assess genotype/phenotype relationships. But in this issue of BioTechniques, a team of researchers led by Jeffrey Marcus (University of Manitoba, Winnipeg, Canada) report on an improved injection needle design that significantly increases transformation efficiencies and experimental outcomes when working with the buckeye butterfly, Junonia coenia. In pilot injections using a non-optimized needle design previously used for germline transformation in another butterfly species, the authors observed 100% mortality of injected Junonia ova. In an effort to find a needle that would work for Junonia transformation, the group took inspiration from standard Drosophila needles, which have a steep taper and large orifice. Employing these “new” needle designs while injecting Junonia ova resulted in a significantly improved outcome: 21.7% of injected ova hatched with a transformation rate of 3%. Marcus and his team next checked whether or not their new design also showed increased transformation efficiency with other butterfly and moth species beyond Junonia—finding that these needles were indeed more efficient than previous designs. The new needles should be a welcome advance for the large number of researchers working on lepidopteran species where higher transformation efficiency is especially important since fewer ova are available, making genetic analysis a more realistic possibility.See “Improved injection needles facilitate germline transformation of the buckeye butterfly Junonia coenia”A new needle design (Needle type 2) for ova injection during germline transformation experiments of butterfly and moth species significantly improved transformation efficiencies.FiguresReferencesRelatedDetails Vol. 56, No. 3 Follow us on social media for the latest updates Metrics Downloaded 122 times History Published online 3 April 2018 Published in print March 2014 Information© 2014 Author(s)PDF download