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To study microRNA function in vivo, the authors optimize lentiviral-driven expression of microRNA target sequences in mice and show dose-dependent inhibition of microRNA-mediated regulation of reporter constructs as well as of natural microRNA targets. With the inhibition of a miR-223, they can phenocopy the knockout of this microRNA.
An efficient pipeline for mapping antibody epitopes is presented. Combining bacterial surface display of peptide libraries, flow cytometric sorting, and pyrosequencing, the approach is amenable to a high-throughput format and should find future application in whole-proteome studies.
A collection of 33,275 human Gateway entry clones and complementary in vitro protein expression methodologies are described that allow proteome-scale production of human proteins. This 'human protein factory' was validated by expression of 13,364 human proteins and assessment of activity in a variety of assays.
Extension of multicolor three-dimensional stochastic optical reconstruction microscopy (STORM) allows super-resolution fluorescence imaging of whole cells and quantitative characterization of subcellular structures and their spatial relationships. This was demonstrated by imaging the entire mitochondrial and tubulin networks in cells.
A simple modification to the optical configuration used for fluorescence photoactivation localization microscopy (FPALM) allows the fluorescence anisotropies of each individual molecule in a nanoscale image to be measured. The method was used to obtain position and orientation information for fluorescently labeled actin or hemagglutinin molecules in fixed fibroblasts.
The combination of a glass window placed on top of a mouse mammary gland with photoswitchable fluorescent protein labeling of implanted tumor cells allows tumor-cell tracking over multiple imaging sessions in orthotopic tumors. Results show the existence of two distinct microenvironments with different tumor-cell invasion and intravasation characteristics.
Co-patterning of a membrane protein bait and a fluorescently labeled prey is used to examine protein-protein interactions in a semiautomated fashion in living cells. Photobleaching experiments and single-molecule imaging further allow dynamic studies of the interaction.
The algorithm Sylamer finds over- or underrepresented nucleotide motifs, such as microRNA seeds, in a gene list ranked according to expression levels and thus establishes whether a microRNA is directly affecting gene expression.
The spatial organization of the genome within the eukaryotic cell nucleus is not random. Automated imaging of thousands of live yeast is now used to build high-resolution probabilistic maps of the locations occupied by individual loci.
New findings challenge the assumption that aggregate genotype data, in which the single-nucleotide polymorphism (SNP) profiles of many people are pooled, conceal the identity of the individuals within that pool.