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In this cellular system, double-strand breaks (DSBs) are induced at I-SceI sites flanked by LacO/TetO arrays. Fluorescently tagged LacR or TetR proteins bind to the arrays and label the DSBs, enabling visualization of chromatin dynamics by time-lapse microscopy.
Genetic techniques in rats are developing apace. Here, Shao et al. describe how to use CRISPR/Cas-mediated genome editing to modify one-cell rat embryos and generate stable rat lines.
Labrijn et al. describe the generation of bispecific antibodies through controlled Fab-arm exchange, involving separate expression of two parental IgG1s containing single matching point mutations, followed by recombination of the 'half molecules'.
Rozier et al. provide a Protocol for whole-mount mRNA in situ hybridization in complex plant tissues. The method is fully compatible with colorimetric or fluorimetric signal detection, including the Tyramide Signal Amplification system.
mGRASP enables the mapping of neuronal circuitry in the mouse brain at multiple scales: at microscale for synapse-by-synapse or neuron-by-neuron analysis, and at mesoscale for revealing local and long-range circuits.
The Gao laboratory provides its protocol for targeted editing of crop genomes using the CRISPR/Cas system. Genome alterations can be induced by transient transfection of rice or wheat protoplasts or by stable transformation of rice plants.
Molecular beacons are dual-labeled oligonucleotides that fluoresce in the presence of complementary mRNA. This protocol describes how to use them to purify specific cell populations by targeting mRNAs that are highly expressed only in the desired cell type.
This protocol anatomically identifies single neurons that have been recorded from freely moving rats, enabling the contribution of individual neuronal types to behavior to be systematically investigated.
Coating of cell culture plates with laminin-521 allows culturing of human pluripotent stem cells and, in combination with E-cadherin, their cloning in individual wells.
This protocol describes how to immunolabel proteins before transmission electron microscopy procedures to ensure ultrastructure and antigen preservation.
Genetic interactions reveal molecular and functional gene networks. Here systematic siRNA-mediated pairwise gene knockdown combined with high-content imaging and computational analysis measures genetic interactions at high throughput in human cells.
MAGE is a rapid in vivo method for introducing mutations into E. coli genomes. Libraries of synthetic ssDNA are used in iterative MAGE cycles to target multiple genomic sites. About five cycles can be carried out by one person in one day.
Colocalization single-molecule spectroscopy (CoSMoS) enables multiple molecules to be simultaneously monitored. This protocol describes how to construct a CoSMoS micromirror total internal reflection fluorescence microscope.
The Isaacs laboratory provides its protocol for CAGE; this method harnesses bacterial conjugation to precisely engineer E. coli genomes to create chimeric strains. A single round of CAGE can be completed in 1 week.
In this protocol, the authors describe deep mutational scanning, an approach that involves selecting for protein function followed by high-throughput DNA sequencing and enables quantification of the activity of protein variants on a massive scale.
The authors of this protocol describe an approach that enables distinguishing de novo–synthesized from pre-existing proteins in the model invertebrate Caenorhabditis elegans, as a way to determine the effect of internal and external stimuli.
Aqueous phase protein-lipid interactions have a role in cell physiology. This protocol describes the analysis of in vivo–assembled protein-lipid complexes in S. cerevisiae using TAP labels, size-exclusion chromatography, SDS-PAGE and HPTLC.
Protoplasts can be used for transient gene expression analysis. This protocol for protoplast isolation from wood-forming tissue will be useful for genetic analysis of woody plants, particularly those that lack methods for stable transformation.