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Nutiu et al. repurpose an Illumina sequencer to quantitatively measure binding affinities between protein and DNA. The data reveal the complex interdependency among binding motif positions and allow improved prediction of gene expression.
Analyzing the dynamics of protein-protein interactions remains a key challenge in systems biology. Bisson et al. combine affinity purification with SRM mass spectrometry to monitor the assembly and dissolution of complexes containing the adaptor protein GRB2 after growth factor stimulation.
Carette et al. use a retroviral gene-trap vector to disrupt and tag >98% of all genes in a haploid human cancer cell line. Cells selected for a phenotype of interest are pooled and characterized by parallel DNA sequencing to circumvent time- and labor-intensive screening of individual clones.
High-throughput synthesis of long DNA molecules would open up new experimental paradigms in synthetic biology and functional genomics. Quan et al. take a step toward this goal by integrating oligonucleotide synthesis, amplification and gene assembly on a single microarray, and apply the technology to optimization of protein translation in a heterologous host.
Much work remains to be done to understand the ‘black box’ of reprogramming, which involves changes in the expression of thousands of genes. Subramanyam et al. identify microRNAs that promote human cell reprogramming and use them to identify key cellular pathways and processes.
It has been extremely challenging to simultaneously improve both the yield and quality of cotton by conventional breeding. Extensive field trials indicate that regulated expression of an auxin biosynthesis gene in the epidermis of cotton ovules improves both the number and fineness of cotton fibers.
Delivery of therapeutic siRNA to specific tissues is a major challenge. Alvarez-Erviti et al. show that exosomes—small vesicles that are naturally secreted by many animal cells—can be engineered to transport siRNA specifically to the brain in mice.
Methods to measure affinities of membrane proteins and soluble ligands are cumbersome and often rely on truncations or other modifications of the membrane protein or ligand. Baksh et al. show that backscattering interferometry is a sensitive and accurate technology for the label-free quantification of ligand–membrane receptor interactions.
Proteins have been largely unexplored as feedstocks for synthesizing fuels and chemicals in microbes, in part because their degradation is not thermodynamically favored in the cell. Huo et al. overcome this by engineering nitrogen flux in E. coli, creating microbes that generate biofuels when grown in protein-rich medium.
Anterior foregut endoderm is the precursor of various tissues that might be amenable to cell replacement therapy, including thymus, thyroid, lung and trachea. Green et al. present a method for generating this endoderm subtype from human embryonic stem cells and induced pluripotent stem cells.
Profiles of biomarker accumlation may sometimes offer more clinically useful information than assays taken at discrete time points. Ling et al. use implantable sensors to show that cumulative measurements of three clinically relevant cardiac biomarkers correlate with infarct size in a mouse model of heart attack.
The inadvertent cutting of nerves is a common adverse event during surgery. To ease visual identification of nerve fibers, Whitney et al. use phage display to develop a peptide that specifically stains peripheral nerves in living mice and in human tissue samples.
TALEs (transcription activator–like effectors) contain a large number of nearly identical repeats, which makes it difficult to synthesize new variants. Feng et al. describe a facile method for assembling TALEs and show TALEs' utility for activating expression of endogenous human genes.
Synthetic biology requires methods for modular, scalable control of gene expression. Liu et al. show that unnatural amino acids can be used to regulate transcription and use the approach to create NOR and OR gates.
Ye et al. mimic a natural pathway for IgG transfer to deliver a vaccine across mucosal barriers. Intranasal immunization of mice with a fusion of a herpes simplex virus type-2 (HSV-2) antigen to an Fc fragment induces long-lasting protection after intravaginal challenge with HSV-2.
Messenger RNA has received little attention as a potential therapeutic agent. Kormann et al. show that intramuscular injection of chemically modified erythropoietin mRNA substantially increases the hematocrit in mice and demonstrate the curative potential of pulmonary mRNA delivery in a mouse model of congenital surfactant protein B deficiency.
Sequencing a human genome using next-generation methods does not distinguish between the two copies of each chromosome. Kitzman et al. determine a haplotype-resolved genome sequence by efficiently constructing and sequencing long-insert clones that cover the diploid genome with a low likelihood of overlap.
Cui et al. generate transgenic rats and mice bearing targeted genomic integrations by enhancing the rate of homologous recombination in single-cell embryos with zinc-finger nucleases. The approach avoids the time-consuming backcrossing involved in generating mutant mice with ES cells and should be applicable to species for which ES cells have not been isolated.
The use of shRNA in vivo to investigate genes involved in proliferation and survival is confounded by the competitive advantage of cells with insufficient shRNA expression. Zuber et al. address this issue with a system that combines Tet-regulated shRNA expression, two fluorescent reporters and robust transactivator production.
Song et al. present the first method for global analysis of 5-hydroxymethylcytosine, a recently identified epigenetic modification in mammalian cells. They use a bacteriophage-derived enzyme to tag the hydroxymethyl group with an azide-modified glucose residue that can be used for affinity purification and sequencing of modified genomic DNA fragments.