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A cardiac-specific reporter genetically engineered into human embryonic stem cells allows the optimization of differentiation protocols and the identification of cell-surface markers—a welcome new tool to help isolate and define cardiac cell lineages.
Retaining the recipient oocyte genome after human somatic cell nuclear transfer permits development to the blastocyst stage and derivation of triploid human embryonic stem cell lines.
In this Perspective the authors discuss strategies for the development of improved fluorescent proteins, with a focus on probes at the red end of the spectrum. They synthesize the literature on chromophore photochemistry and protein structure to identify residues for targeted mutagenesis, and consider improvements in molecular evolution methodologies to enable improved screening for desired probes.
Mutations at arbitrarily sampled genomic positions are identified using next-generation sequencing and are used to infer the lineage of DNA damage–prone 'mutator' mouse cells in culture.
The microbial rhodopsin protein, Archaerhodopsin 3, can function as a rapid and highly sensitive genetically encoded voltage indicator in mammalian cells that is capable of detecting single action potentials with a signal-to-noise ratio greater than 10. A mutant lacking proton pumping displays greater sensitivity but a slowed response.
The DNA modification 5-hydroxymethylcytosine has recently been implicated in several biological processes. Enrichment by selective chemical labeling in combination with single-molecule, real-time sequencing provides sensitive detection of this epigenetic mark in genomic DNA at base-pair resolution.
Unique molecular identifiers (UMIs) associate distinct sequences with every DNA or RNA molecule and can be counted after amplification to quantify molecules in the original sample. Using UMIs, the authors obtain a digital karyotype of an individual with Down's syndrome and quantify mRNA in Drosophila melanogaster cells.
Conjugation of triplet-state quenchers to the small organic cyanine fluorophore, Cy5, increases photostability without affecting its spectral characteristics. This allows longer fluorescence imaging with a concomitant reduction in blinking both in vitro and in living cells.
Presented is a study of gene regulation during development using a combination of chromatin immunoprecipitation and high-throughput sequencing (ChIP-seq) and directed differentiation of mouse embryonic stem cells inducibly expressing epitope-tagged transcription factors.
An empirical approach for identifying optimal proteotypic peptides and fragmentation patterns from in vitro–synthesized proteins, for targeted proteomics applications, is described.
Molecular engineering allows stoichiometric and co-localized expression of two optogenetic actuators, spaced by a fluorescent protein and an additional transmembrane helix in a single protein fusion. The method provides modular optogenetic tools for bidirectional membrane potential control or synergistic effects on neuronal activity.
A quantitative characterization of the switching properties of 26 organic dyes relates these properties to the quality of localization-based super-resolution images they generate. The data are a useful resource for selecting dyes and point to avenues for future analysis.
A quantitative proteomics approach to characterize protein palmitoylation dynamics on a global scale in cells, as well as to identify enzymes responsible for the regulation of palmitoylation, is described.
