In the Spotlight

For nearly a decade, researchers have been developing methods to regulate gene expression by literally flipping a switch, (i.e., activating a molecule through light exposure). Early efforts used a strategically placed caging group on small-molecule inducers of gene expression like doxycycline. In such systems, the modified compounds were blocked from protein interactions until UV-induced uncaging. A downside is the risk of diffusion of the inducers, which might lead to activation of non-target cells. An alternative is to cage a critical residue of an enzyme, which is the approach taken by Edwards et al. in a report in ACS Chemical Biology describing a new light-activated Cre recombinase. Cre/lox P recombination is frequently used for knocking out or modulating gene expression, but spatial and temporal control of its activity has been elusive. Using an established bacterial expression system that relies on an unnatural tRNA to insert a caged tyrosine at a specific site, the authors produced a supply of temporarily inactivated recombinase. To test whether the caged Cre could be reactivated, the authors cotransfected it with the Cre-Stoplight plasmid, which contains a lox-bounded DsRed and terminator upstream of GFP. In the presence of wild-type Cre, recombination occurs and the cell fluoresces green. When provided with caged Cre, the cells are red, indicating no recombinase activity. After UV irradiation, GFP appears, confirming that the modified Cre can be reactivated, a result that gives a green light to applications requiring fine control of recombination.

Edwards et al. Light-activated Cre recombinase as a tool for the spatial and temporal control of gene function in mammalian cells. ACS Chem Biol. [Epub ahead of print, May 18, 2009; doi: 10.1021/cb900041s].

An Inside Job

Coaxing primary cells to take up siRNAs is a tough task, but a report from Steven Dowdy and colleagues in Nature Biotechnology offers an opening into this vexing problem. Dowdy is known for protein transduction, which uses positively charged cell-penetrating peptides, as from HIV-1 Tat and Drosophila Antennapedia. Previous attempts to conjugate peptide transduction domains (PTDs) to siRNAs flopped because of aggregation. The new report shows that the key is fusing the PTD to a double-stranded RNA binding domain (DRBD). In initial tests in a lung adenocarcinoma cell line, the method elicited better knockdown and less cytotoxicity than lipofection. Flow cytometry showed a population-wide response to siRNA transduced using PTD-DRBD, while roughly 20% of cells undergoing lipofection weren't silenced. Cells in which siRNA had been introduced with PTD-DRBD also responded quickly and had few off-target effects. When the authors tested PTD-DRBD–mediated silencing in hard-to-transfect T cells, they showed that the method outperformed lipofection in both Jurkats and primary murine T cells. There were equally encouraging results for human umbilical vein endothelial cells (desirable for use in RNAi screens) and human embryonic stem cells. To gauge the method's prospects for delivery of RNAi therapeutics in vivo, the authors treated luciferase-expressing mice intranasally with PTD-DRBD–delivered siRNAs, and showed a greater than 50% knockdown of luciferase level in the nasal and tracheal passages. Though the in vivo data are preliminary, the mix of RNAi targets and cell types used indicate that PTD-DRBD–induced transduction is a robust way to deliver siRNAs inside cell types recalcitrant to existing methods.

Eguchi et al. 2009. Efficient siRNA delivery into primary cells by a peptide transduction domain-dsRNA binding domain fusion protein. Nat Biotechnol 27(6):567–571.

Dump the Pump

Captivatingly tiny microfluidics devices are often tethered to something far more prosaic—a pump. Besides the inconvenience of needing external equipment for running assays, flow-based systems require valves, which can be complicated and expensive to engineer at the microscale. By contrast, microplate systems are easy to make and multiplex, but present challenges for dispensing and storing small volumes in wells. In Lab on a Chip, Ismagilov and colleagues describe an attractive solution they call SlipChip. The device consists of two glass plates, one etched with a set of reagent wells, and the other with an offset series of sample wells. Before the plates are brought together, the reagent wells are loaded using Ismagilov's previously described plug-based method. In brief, tiny bubbles of reactant are ‘parked’ within an inert carrier fluid contained in a Teflon tube; with a micromanipulator-controlled syringe, each plug is dispensed into a separate well. Because the device is immersed in the carrier, the tiny volumes do not evaporate (and can be stored). When the device is assembled, the wells from the top plate align with a channel in the bottom plate, creating a trough for adding sample. Then, by sliding the top plate over the bottom plate, the sample wells are pushed over the reagent wells, thus mixing reagent and sample. Despite its simplicity, SlipChip allows reproducible filling and mixing, and suffers minimal cross-contamination. As an example application, the authors tested SlipChip in a protein crystallization screen. Each reagent well was loaded with a different buffer, and sample wells were filled with a 1-µL aliquot of protein solution. Despite the variation in buffer properties, the device worked well and crystals appeared in the expected wells. SlipChip should be attractive whenever small quantities of a sample must be exposed to many reagents in parallel, and its lack of attached hardware will be of particular interest for point-of-care or field-based sensing applications.

Du et al. SlipChip. Lab Chip [Epub ahead of print, May 15, 2009; doi: 10.1039/b908978k].