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An array-based high-throughput approach termed Escherichia coli synthetic genetic array, or eSGA, now allows comprehensive genetic interaction screens in bacteria. The method makes use of bacterial conjugation and robotic technology to generate double mutants on a genome-wide scale. In this issue, another paper presents GIANT-coli, a very similar approach.
An array-based high-throughput approach, genetic interaction analysis technology for Escherichia coli (GIANT-coli), now allows comprehensive genetic interaction screens in bacteria. The method uses bacterial conjugation and robotic technology to generate double mutants on a genome-wide scale. In this issue another paper presents eSGA, a very similar approach.
Cell-cell coupling via gap junctions has been extensively studied in vitro and in heterologous systems, but in vivo studies are still few. A new class of photoactivatable bioconjugates is now used to monitor gap junctional coupling in living Caenorhabditis elegans.
In vitro studies of neuronal function have mainly been limited to two-dimensional networks of cultured neurons. Use of transparent colloids as a moveable support for neuronal growth allows user-guided construction of optically accessible three-dimensional networks whose function can be manipulated and measured.
Single-particle tracking methods allow detailed analysis of protein movement in cells, but existing tracking algorithms have substantial limitations, particularly at high particle densities. A new software tool overcomes some of these limitations and is used to track CD36 receptors and assay the lifetime of clathrin-coated pits. Also in this issue, Sergé et al. describe an alternative software tool for high-density single-particle tracking.
Designing fluorescent protein-based sensors that display large changes in fluorescence resonance energy transfer (FRET) is challenging. Redesign of a FRET-based voltage sensor using new fluorescent proteins increased the sensor response to changes in membrane voltage and measurements at warmer temperatures displayed faster kinetics comparable to action potentials.
A library of universal Saccharomyces cerevisiae Barcoder strains for efficient tagging is presented. It is used to tag a collection of hypomorphic alleles of essential yeast genes and applied to chemical genetic screens. Also in this issue, Breslow et al. present a similar collection of hypomorphic alleles, coupled with a sensitive growth assay for improved genetic interaction studies.
To increase the range and precision of genetic interaction studies in Saccharomyces cerevisiae, a collection of hypomorphic alleles of essential yeast genes and a highly sensitive flow cytometry–based growth competition assay are presented. Also in this issue, Yan et al. present a similar strain collection, tagged with unique bar-code identifiers, and use this collection in pooled chemical genetic screens.
A new base caller for the Illumina Genome Analyzer uses machine learning to compensate for noise factors and improves accuracy for up to 78-base-pair sequencing reads.
Single-particle tracking methods allow detailed analysis of protein movement in cells, but existing tracking algorithms have substantial limitations, particularly at high particle densities. A new software tool overcomes some of these limitations and can be used to track high-density particles in cell membranes. Also in this issue, Jaqaman et al. describe an alternative software tool for high-density single-particle tracking.
Automated imaging of the Caenorhabditis elegans embryo now allows monitoring of the timing and relative expression of individual reporter genes at single-cell resolution over almost all of embryonic development. Future systematic analysis could be used to reveal gene expression patterns of every cell during development.
Many proteins, including G protein–coupled receptors (GPCRs), interact to form oligomers at the cell surface. A combination of bioluminescence resonance energy transfer (BRET) and fluorescence resonance energy transfer (FRET) in a technique called sequential resonance energy transfer (SRET) extends these methods to study higher-order oligomers of GPCRs or other proteins.
A major bottleneck for genetic approaches in model organisms is the application of state-of-the-art technologies to phenotyping. Now, using a microfluidic chip, high-resolution imaging of fluorescent reporters and accurate sorting is demonstrated in an automated manner in Caenorhabditis elegans.
Cells in vivo are exposed not only to soluble factors but also to immobilized ligands. Controlled immobilization of functional growth factors yields dose-dependent responses in mouse embryonic stem cells in vitro and allows the effects of immobilized versus soluble ligands to be studied.
The complete set of coding sequences, including all splice isoforms, is not known for any metazoan organism. Combination of a normalized pooling scheme and a new assembly algorithm with 454 sequencing yields a methodological pipeline for isoform discovery. The validated pipeline may now be applied genome-wide.
Conventional techniques for generating transgenic mice are quite costly, require substantial resources and necessitate killing the mouse. In contrast, in vivo electroporation of repopulating spermatogonial cells in the mouse testis can produce male mice for siring multiple distinctive transgenic founders for over a year.
Current approaches for live imaging of cellular actin dynamics have several drawbacks. Now the use of Lifeact, a 17-aa actin-binding peptide from yeast that is not present in higher eukaryotes, allows imaging of actin dynamics in live mammalian cells without disruption of function and without competition with endogenous binding proteins.
A combination of improved in vitro embryo culture and optical projection tomography allows development of the mouse limb bud to be monitored over time. Developmental changes seen in vitro are benchmarked against in vivo development, and tissue movements are quantitatively described.
The mouse transcriptome in three tissue types has been analyzed using Illumina next-generation sequencing technology. This quantitative RNA-Seq methodology has been used for expression analysis and splice isoform discovery and to confirm or extend reference gene models. Also in this issue, another paper reports application of the ABI SOLiD technology to sequence the transcriptome in mouse embryonic stem cells.
Application of next-generation sequencing using the ABI SOLiD technology to mammalian transcriptome analysis enabled a survey of the content, the complexity and the developmental dynamics of the embryonic stem cell transcriptome in the mouse. Also in this issue, Mortazavi et al. report Illumina technology–based RNA-Seq analysis of the mouse transcriptome in three different tissues.