Ever since Sydney Brenner plucked C. elegans from obscurity, this otherwise forgettable nematode has been an indispensable tool in the hands of developmental biologists, neuroscientists, and biology-of-aging researchers, among others. Of course, few who work with this model organism today would want to engage in the sort of painstaking labor epitomized by John Sulston's famous elegans fate map. These days, it's all about throughput, a fact not lost upon George Whitesides and colleagues, who describe a microfluidic device designed to hold 100+ worms in place for side-by-side imaging and manipulation. It's not hard to see why this contraption holds real advantages over previous methods for immobilizing worms, such as the use of glue or paralytic compounds. The new approach can be thought of as multiplexed worm trapping: the nematodes are swept down channels that taper down until the worms are straightjacketed in place. Without room to engage in the sinusoidal motions that normally propel them, the worms are helpless until the flow direction is reversed and the animals are swept back into channels generous enough for maneuvering. The authors show that worms can easily be loaded into the apparatus, and are trapped in positions that permit high-resolution imaging of structures likely to be of interest during experimentation, including the pharynx, mouth, and developing embryos. In addition, they demonstrate that worms that are trapped and subsequently released are indistinguishable from control animals in terms of behavior, lifespan, and reproductive capacity—certainly not a claim that can be made for the gluing method of worm immobilization. The authors envision that this device would be well suited for phenotypic scoring during genetic screens. In addition, the device could likely be customized to subject trapped worms to a variety of experimental manipulations, including laser ablation and exposure to chemical, mechanical, or electrical stimuli.
Hulme et al. 2007. A microfabricated array of clamps for immobilizing and imaging C. elegans. Lab on a Chip [Epub ahead of print, August 16, 2007].
Acid TestHighly parallelized analysis of the effects of compound libraries on ion channels represents an important investigative modality in chemical biology and drug discovery. One method for high-throughput screening employs voltage-sensitive dyes. Oxonol is frequently used as a FRET acceptor, participating in producing a signal as it redistributes and interacts with a hydroxycoumarin-based FRET donor placed at the outer cell membrane. Optical signal capture provides a high-throughput means of screening for ion channel function, provided that the ambient pH is restricted to physiological conditions. However, some experiments necessitate a lower pH, as in analysis of acid-sensing ion channels (ASICs), which are a family of proteins that appear to participate in signalling related to pain, mechanosensation, and neurotransmission. ASICs have pKa values ranging from 4.5 to 6.5, which makes them vulnerable to pH-induced artifacts when using the standard FRET pairs. Finding a suitable pH-insensitive replacement for hydroxycoumarin is not trivial: in addition to being less vulnerable to acidic conditions, potential FRET donors must also persist on the outer face of the cell membrane, resist aggregation, and exhibit suitable excitation and emission wavelengths. In a recent report in the Journal of Biomolecular Screening, Maher et al,. report that certain pyrenetrisulfonate dyes can meet these criteria, serving as FRET donors that are almost as effective as the standard coumarin-linked phospholipid donor. The authors provide detailed physicochemical parameters of the dyes they examined and show that the best could be used in a standard high-throughput screening pipeline employing an optical assay system in scans of HEK-293 cells grown in 384-well plates.
Maher et al. 2007. pH-insensitive FRET voltage dyes. Journal of Biomolecular Screening [Epub ahead of print, May 21, 2007].
Promotional PlanWhen it comes to marketing, successful promoters know that one size does not fit all: sometimes the highest rewards arise by tuning the message to match each market sector. Although most scientists aren't particularly market-savvy, they certainly do recognize the value of a well-suited promoter—gene promoter, that is. After all, a strong promoter in one cell type might be a dud in another. Despite the critical implications of this variability, collecting such data can be a frustrating step in a research project, particularly in such a fast-paced field as stem cell research. Fortunately, in a paper appearing in Molecular Therapy, Hong et al,. describe their characterization of promoter behavior in mouse embryonic stem cells. Promoter choice is significant in stem cells because many therapeutic uses would require high-level expression of a gene of interest. A popular way to introduce expression cassettes is through lentiviral vectors, and so Hong et al. prepared a suite of HIV-based vectors that differed in the choice of promoter used to drive expression of a GFP marker. They then compared transduction efficiencies and expression levels in ES cells that had been brought to different stages of differentiation. They found that the elongation factor 1α (EF1α) promoter gave the best results in the initial ES cells and embryoid body cells. However, as the cells became more differentiated, the activity of the CMV promoter rose rapidly, and other constructs, particularly those containing a CMV-β-actin hybrid promoter or the phosphoglycerate kinase 1 promoter, showed enhanced expression. Because persistence of expression is likely to be important in any therapeutic use of ES cells, the authors analyzed expression level over time and during differentiation. Cells maintained in an embryonic state showed no detectable decrease in GFP level over a 60-day period. In the vector containing the EF1α promoter, GFP transcripts declined by 40% in ES cells that were differentiated to embryoid bodies and then maintained for four weeks. Further differentiation to neural precursors and fully differentiated cells caused dramatic declines in expression level. These results confirm the utility of pseudotyped lentiviral vectors for transduction of ES cells and their derivatives, but suggest that promoters intended for use in ES cells be carefully selected according to the differentation “destination.”
Hong et al. 2007. Functional analysis of various promoters in lentiviral vectors at different stages of in vitro differentiation of mouse embryonic stem cells. Molecular Therapy 19:1630-1639.
Battering RAMNot all bacteria are as obliging as E. coli when it comes to mutagenesis. For example, most members of genus Clostridium—including the notorious C. botulinum-obstinately resist standard techniques for insertional inactivation of genes. Given that various Clostridium species have relevance to pathogenesis, biofuels, and cancer therapy, this recalcitrance represents a significant impediment to characterization and engineering of these organisms. Heap et al,. speculated that a solution might exist in the adaptation of a group II intron retrohoming construct containing a retrotransposition-activated marker (RAM) element. The mobile group II intron of Lactococcus lactis mediates insertion into specific DNA locations by sequence recognition between the excised intron lariat and the target DNA. Altering the intronic sequences allows targeted insertion. The RAM element, in turn, permits screening of insertional mutants. It consists of an antibiotic resistance gene that is maintained in an inactive state by an interrupting, self-splicing group I intron. In this way, the antibiotic resistance gene is not active in the plasmid used to initiate the retrohoming-mediated insertional mutagenesis procedure, making its expression a reliable marker of successful integration. Heap et al. describe the construction of a shuttle plasmid allowing inducible expression of the group II intron-containing transcript and a nested RAM element conferring erythromycin resistance. They show that they can derive stable mutants in a variety of Clostridium species, including those in which genes have never been successfully inactivated before. This development should allow harnessing of existing sequence information for functional genomics.
Heap et al. 2007. The ClosTron: A universal gene knock-out system for the genus Clostridium. Journal of Microbiological Methods 70:452-464.
