BioTechniquesVol. 60, No. 1 BioSpotlight / CitationsOpen AccessBioSpotlight / CitationsNathan S. Blow & Nijsje DormanNathan S. BlowSearch for more papers by this author & Nijsje DormanSearch for more papers by this authorPublished Online:16 Mar 2018https://doi.org/10.2144/000114366AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInReddit Pigmentation to the rescue in zebrafishTransgenic animals play a key role in the quest to elucidate gene function. Zebrafish, in particular, have emerged as key transgenic models for genetic screens and drug discovery experiments. Yet, screening and sorting for transgene expression in zebrafish can be both timeconsuming and costly. In this issue of BioTechniques, Hui Feng and colleagues at the Dana-Farber Cancer Institute describe an efficient transgenesis strategy utilizing pigmentation rescue for identification of stable transgenic fish. To create an easy-to-assess transgene marker, the authors turned to nacre mutants, which possess a single change in the mitfa gene resulting in a lack of melanophores during development, thus producing fish without pigmentation and striping along their sides. Using an I-SceI meganuclease approach, Feng and colleagues designed their transgenesis strategy to reverse the mitfa mutation and rescue the pigmentation phenotype as an indication of expression of the transgene of interest. Using the approach, they were able to quickly assess both the complete and mosaic rescue phenotypes by sight alone, enabling speedier identification of zebrafish carrying the transgene.See “Efficient transgenesis mediated by pigmentation rescue in zebrafish”Milk collectorMaternal milk provides a strong source of nutrients to offspring while also decreasing the risk of diseases such as obesity and hypertension. Studying the composition of maternal milk and its overall effect on offspring health and development is therefore an important area of research. When using mice as an experimental model, scientists often need to stimulate milk production in the mother through injection of oxytocin. However, these injections have the potential to modify milk composition or alter the behavior of the mother. In this issue of BioTechniques, Pierre Fafournoux and his colleagues at the INRA Human Nutrition Unit in France describe a better methodology for inducing milk production in mice. By naturally stimulating the mammary gland of the mother following reunion with her pups, the researchers were able to collect significant amounts of mouse milk, without the need for oxytocin injections. This approach, which is both less invasive and more cost-effective, will enhance the study of milk composition during important physiological time points and in response to crucial interactions between mother and offspring.See “Method for collecting mouse milk without exogenous oxytocin stimulation”Protein localization via electron microscopyThe idea of “systems” electron microscopy (EM) for analyzing protein distribution may seem absurd, considering the time and labor requirements of EM. Replacing immunogold labeling with APEX (ascorbate peroxidase) tagging—which under appropriate conditions can produce an electron-dense stain—simplifies EM-resolution protein localization, but each protein of interest must be cloned in fusion with the APEX tag. Now, as a promising step toward rapid EM-based screening, Ariotti et al. describe an APEX—GFP nanobody expression construct for use with clone libraries tagged with GFP or YFP. In a test of GFP-tagged phosphoinositide variants cotransfected with the APEX fusion, the authors procured localization data for 8 proteins in just 3 days. Resolution is ∼10 nm, and quantitative analyses of density enrichment are possible. The strategy also works in vivo, as shown by protein localization in zebrafish produced by crossing animals expressing the APEX fusion with existing GFP-tagged strains.N. Ariotti. 2015. Modular detection of GFP-labeled proteins for rapid screening by electron microscopy in cells and organisms. Dev Cell. 35:513-25.Nucleic acid—nanoparticle transfectionSpherical nucleic acid (SNA)—nanoparticle conjugates have proven effective at delivering nucleic acids to a variety of cell types through caveolae-mediated endocytosis. These conjugates consist of a dense shell of oligonucleotides surrounding a core that can consist of quantum dots, gold nanoparticles, or liposomes. This range of formats inspired Brodin et al. to examine whether a protein could form the nanoparticle core, creating a “ProSNA”. As their test case, the authors took the homotetrameric enzyme β-galactosidase (β-gal ) and to its surface attached some 25 G-rich oligonucleotides via an azide-containing linker. Although standard β-gal cannot traverse membranes, cellular uptake of fluorescently labeled ProSNA β-gal was readily apparent by flow cytometry, even at subnanomolar protein concentrations. What's more, tests with chromogenic and fluorogenic substrates showed the enzyme retained its catalytic activity after internalization. Given the absence of visible toxicity, ProSNAs appear to be a promising new vehicle for protein transfection.J.D. Brodin. 2015. DNA-mediated cellular delivery of functional enzymes. J Am Chem Soc. 137:14838-41.FiguresReferencesRelatedDetails Vol. 60, No. 1 Follow us on social media for the latest updates Metrics History Published online 16 March 2018 Published in print January 2016 Information© 2016 Author(s)PDF download
Read full abstract
Aug 3, 2024
José Felipe Orzuna-Orzuna + 7
Open Access
