Research articles

Upon binding multiple fluorophores and being complexed into tetramers, these RNA imaging probes show high sensitivity and can detect single endogenous RNA molecules at low probe concentration.

A fluorescent protein, Sirius, with the most blue-shifted emission spectrum to date, is reported. Sirius allows extended multicolor imaging as well as imaging in acidic environments owing to its pH insensitivity.

Previous whole-transcriptome analysis by RNA-Seq required hundreds of thousands of cells or microgram amounts of RNA. A modification of the cDNA library preparation method now allows unbiased capture of the majority of genes expressed in a single blastomere and oocyte. cDNA sequencing on the SOLiD platform facilitates the quantitative analysis of the transcriptome complexity in a single cell.

piggyBac transposons carrying reprogramming factors are used to reprogram mouse embryonic fibroblasts, with efficiencies equivalent to retroviral transduction, and then removed from the induced pluripotent state cell genome without a trace.

As an alternative to the use of radioactively labeled amino acids, incorporation of puromycin into proteins allows evaluation of translation in heterogenous cell populations by flow cytometry analysis after staining with an antibody to puromycin.

Dense mapping of DNase I cleavage sites across the whole yeast genome by next-generation sequencing reveals a global view of the binding of regulatory proteins to genomic DNA. The high resolution allows the identification of new binding sites for known factors as well as the de novo derivation of factor binding motifs.

The PCR step in the preparation of sequencing libraries for the Illumina Genome Analyzer can introduce coverage bias, especially in very (A+T)-rich genomes. By directly annealing template DNA to adapters with sequences needed for attachment in the flow cell, PCR can be omitted as cluster amplification in the flow cell enriches for fully ligated templates.

Long-term engraftment of hematopoietic stem cells into the bone marrow of a recipient depends on immunological compatibility between donor and host, or ablation of the host's immune system by irradiation. A 'universal recipient' mouse model now shows that mice that lack T, B and NK cells and bear mutations in the tyrosine kinase Kit accept any donor HSC without irradiation.

Rats are desensitized to xenografts of human neural or embryonic stem cell–derived cells by exposure to the xenogeneic cells during the neonatal period. Brain grafts survive in immunocompetent rats without chronic immunosuppression, allowing long-term studies.

An automated system to measure aggression and courtship in pairs of interacting Drosophila is presented and should allow large-scale screens of these behaviors in the future.

The base-calling algorithm SNPSeeker detects single-nucleotide polymorphisms with frequencies that are below the error rate of the sequencing platform. It is thus well suited to analyze data from large pooled samples and find rare variants that may contribute to diseases or complex traits.

Complex three-dimensional structures on cellular surfaces are often damaged during high-resolution imaging of live cells. Now, hopping probe scanning ion conductance microscopy—which uses a hopping nanopipette that 'hops' instead of 'sliding'—protects surface structures from probe-induced damage.

Sialic acid–containing cell-surface glycoproteins can be chemically labeled with a biotin tag under mild conditions. The method is highly efficient and uses commercially available reagents; it should be useful for studying glycoprotein trafficking as well as in glycoproteomics applications.

Dual-color fluorescence recovery after photobleaching (FRAP) is used to investigate dimerization and higher-order complex formation of receptors at the surface of live cells. A defined fraction of receptors is immobilized with antibodies, and the mobility of the nonimmobilized fraction is measured by FRAP.

Optical stimulation of channelrhodopsin-2 expressed in neurons of the motor cortex is combined with electromyogram recordings or motion-sensing of limb muscles to achieve fast motor mapping in the mouse.

A map of single nucleotide polymorphisms (SNPs), also called a haplotype map, is very informative for mapping complex trait loci, but obtaining haplotypes over long genomic distances is very challenging. The combination of dye-labeling each SNP on PCR fragments with total internal reflection microscopy will allow the reading of long-range haplotypes with relative ease.

Solid-state NMR spectroscopy is used to elucidate structural details about proteins that cannot be easily studied by X-ray crystallography, but because the technique is not very sensitive, large sample amounts are required, limiting its biological application. A combination of optimizations now increases the sensitivity of solid-state NMR spectroscopy by up to 5-fold.

Absolute quantitative information about the stoichiometry of protein complex components can be obtained with a modified affinity purification–mass spectrometry method, as demonstrated for the human protein phosphatase 2A network.

As cells move over a substrate, they need to first sever their contact with the matrix by detaching focal adhesions. A setup that allows spatially and temporally controlled release of focal adhesions now facilitates the quantitative measurement of cell movement across a substrate.

An improved version of the green-to-red photoconverting EosPF is presented. mEos2 is a functional fusion partner for many cellular proteins and retains the high localization precision of EosFP in super-resolution imaging. Also in this issue, Subach et al. present an inducible mCherry variant for super-resolution imaging.