The C. elegans model system excels for studies as diverse as aging research, genetic screens for identifying signaling pathways, and even maze-based tests of learning. However, worms must be at the same developmental stage, since age-dependent variation can obscure results. Traditionally, synchronized populations are prepared by bleaching egg-containing hermaphrodites: this treatment kills adults and liberates the eggs, which are then hatched into a synchronized culture. Unfortunately, this procedure is not convenient for preparing large numbers of worms; instead, in Lab on a Chip, Rezai et al. show how to let the worms sort themselves. The impetus is a direct current, which the authors have previously shown can spur worms to swim toward the cathode. This electrotaxis requires a certain range of voltage: below the lower threshold the animals swim randomly; above the upper threshold the worms become unable to control their movements. Because older worms are more sensitive to electric fields, Rezai et al fabricated a device consisting of two chambers separated by parallel electric traps (narrowed portions of the connecting channels in which the electric field is enhanced). The worms are loaded at the anode side and the electrical field is set so the strength within the traps selectively paralyzes the older animals. While older worms accumulate in the loading chamber, younger, less-affected animals transit to the cathode chamber. The authors were easily able to separate young adults from stage 3 or 4 larvae. Once sorted, the worms seem unaffected by their treatment, showing normal chemotactic responses and lifespan. It is even possible to use the device to sort young and old adults, though with lower purity than sorts of larvae versus adults. The authors envision improvements to throughput, but even as described the apparatus sorts nearly 80 worms per minute. The resulting synchronized populations should be useful for mutant screening and could be used as input in subsequent rounds of sorting for more demanding applications.
P. Rezai et al. Electrical sorting of Caenorhabditis elegans. Lab Chip. [Epub ahead of print, March 30, 2012; doi:10.1039 /c2lc20967e].Control Freak
Knock-in and knockout mice have proved a functional genomics treasure trove. However, transgenic approaches fall short in terms of dictating relative protein amount. The ability to dial protein expression level up or down would enable analysis of complex, heavily regulated pathways such as those involved in metabolism. This premise has inspired a new strategy, described by Rodriquez and Wolfgang in Chemistry & Biology, for combined chemical-genetic regulation of posttranslational stabilization. As proof of principle, the authors targeted a decarboxylase that acts on a key compound in fatty acid metabolism. To regulate protein stability, they used the FK506 binding protein (FK506BP) destabilization domain and its stabilizing ligand Shield-1. Proteins expressed as FK506BP fusions are degraded in the absence of Shield-1, but are stabilized in a dose-dependent manner by the cell-permeant peptide. In the vectors created by Rodriguez and Wolfgang, decarboxylase is expressed as a carboxy-terminal fusion with FK506BP-YFP, after which an mCherry marker was placed downstream of an internal ribosome entry sequence. The YFP monitors Shield-1-regulation, while the mCherry is a constitutively expressed control. For use in mice, this cassette was incorporated downstream of a vector containing a floxed sequence of stop codons. The flanking lox sequences are lox2272, preventing inadvertent recombination with loxP sequences in conditional knockout mice. Activation of the FK506BP fusion cassette can be controlled through the appropriate choice of transgenic mice with inducible or tissue-specific promoters driving Cre recombinase. A key innovation of the new methodology, however, is the second layer of control exerted by Shield-1-dependence. As one example, the authors describe a cross with mice transgenic for tamoxifen-inducible Cre recombinase. Cre expression is dormant until intraperitoneal tamoxifen injection, but the decarboxylase-YFP fusion remains undetectable until a new series of intraperitoneal injections, this time with Shield-1. After the dual controls are lifted, there is a ~3-fold increase in fatty acid oxidation compared to control mice. In a study aimed at examining tissue-specific effects of changes caused by metabolic pathways, the authors also tested a cross involving mice in which the Cre recombinase was controlled by a muscle-specific promoter. In the progeny, the recombinant decarboxylase was expressed in muscle tissue in a manner responsive to the dose of Shield-1. The authors envision extending this strategy to mice where the gene of interest has been knocked out: the protein could be restored in wild-type or mutant form with timing and/or amount exactly dictated by the experimenter's needs. Since Shield-1-mediated control is fully reversible, this chemical-genetic strategy has broad scope for discerning protein function and pathway regulation.
S. Rodriguez and M.J. Wolfgang. 2012. Targeted chemical-genetic regulation of protein stability in vivo. Chem Biol. 19:391-8.